Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders. PMID:21533138

Background Retroelements can successfully colonize eukaryotic genome through RNA-mediated transposition, and are considered to be some of the major mediators of genome size. The migratory locust Locusta migratoria is an insect with a large genome size, and its genome is probably subject to the proliferation of retroelements. An analysis of deep-sequencing transcriptome data will elucidate the structure, diversity and expression characteristics of retroelements. Results We performed a de novo assembly from deep sequencing RNA-seq data and identified 105 retroelements in the locust transcriptome. Phylogenetic analysis of reverse transcriptase sequences revealed 1 copia, 1 BEL, 8 gypsy and 23 non-long terminal repeat (LTR) retroelements in the locust transcriptome. A novel approach was developed to identify full-length LTR retroelements. A total of 5 full-length LTR retroelements and 2 full-length non-LTR retroelements that contained complete structures for retrotransposition were identified. Structural analysis indicated that all these retroelements may have been activated or deprived of retrotransposition activities very recently. Expression profiling analysis revealed that the retroelements exhibited a unique expression pattern at the egg stage and showed differential expression profiles between the solitarious and gregarious phases at the fifth instar and adult stage. Conclusion We hereby present the first de novo transcriptomeanalysis of retroelements in a species whose genome is not available. This work contributes to a comprehensive understanding of the landscape of retroelements in the locust transcriptome. More importantly, the results reveal that non-LTR retroelements are abundant and diverse in the locust transcriptome. PMID:22792363

Cucumber gray mold caused by Botrytis cinerea is considered one of the most serious cucumber diseases. With the advent of Hi-seq technology, it is possible to study the plant–pathogen interaction at the transcriptome level. To the best of our knowledge, this is the first application of RNA-seq to identify cucumber and B. cinerea differentially expressed genes (DEGs) before and after the plant–pathogen interaction. In total, 248,908,688 raw reads were generated; after removing low-quality reads and those containing adapter and poly-N, 238,341,648 clean reads remained to map the reference genome. There were 3,512 cucumber DEGs and 1,735 B. cinerea DEGs. GO enrichment and KEGG enrichment analysis were performed on these DEGs to study the interaction between cucumber and B. cinerea. To verify the reliability and accuracy of our transcriptome data, 5 cucumber DEGs and 5 B. cinerea DEGs were chosen for RT-PCR verification. This is the first systematic transcriptomeanalysis of components related to the B. cinerea–cucumber interaction. Functional genes and putative pathways identified herein will increase our understanding of the mechanism of the pathogen–host interaction. PMID:26536465

Background After 10-year-use of AFLP (Amplified Fragment Length Polymorphism) technology for DNA fingerprinting and mRNA profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. AFLP marker systems have been and are being extensively exploited for genome scanning and gene mapping, as well as cDNA-AFLP for transcriptome profiling and differentially expressed gene cloning. The evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. This may be achieved by means of the Gene Ontology (GO), consisting in three structured vocabularies (i.e. ontologies) describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. In this paper, the functional annotation of about 8,000 AFLP-derived ESTs retrieved in the NCBI databases was carried out by using GO terminology. Results Descriptive statistics on the type, size and nature of gene sequences obtained by means of AFLP technology were calculated. The gene products associated with mRNA transcripts were then classified according to the three main GO vocabularies. A comparison of the functional content of cDNA-AFLP records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ESTs of Arabidopsis and rice, respectively. On the whole, the statistical parameters adopted for the in silico AFLP-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable GO results. Such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species. Conclusion Reliable GO annotations of AFLP-derived sequences can be gathered through the optimization of the experimental steps

Background Artemisia sphaerocephala, a semi-shrub belonging to the Artemisia genus of the Compositae family, is an important pioneer plant that inhabits moving and semi-stable sand dunes in the deserts and steppes of northwest and north-central China. It is very resilient in extreme environments. Additionally, its seeds have excellent nutritional value, and the abundant lipids and polysaccharides in the seeds make this plant a potential valuable source of bio-energy. However, partly due to the scarcity of genetic information, the genetic mechanisms controlling the traits and environmental adaptation capacity of A. sphaerocephala are unknown. Results Here, we present the first in-depth transcriptomicanalysis of A. sphaerocephala. To maximize the representation of conditional transcripts, mRNA was obtained from 17 samples, including living tissues of desert-growing A. sphaerocephala, seeds germinated in the laboratory, and calli subjected to no stress (control) and high and low temperature, high and low osmotic, and salt stresses. De novo transcriptome assembly performed using an Illumina HiSeq 2500 platform resulted in the generation of 68,373 unigenes. We analyzed the key genes involved in the unsaturated fatty acid synthesis pathway and identified 26 A. sphaerocephala fad2 genes, which is the largest fad2 gene family reported to date. Furthermore, a set of genes responsible for resistance to extreme temperatures, salt, drought and a combination of stresses was identified. Conclusion The present work provides abundant genomic information for functional dissection of the important traits of A. sphaerocephala and contributes to the current understanding of molecular adaptive mechanisms of A. sphaerocephala in the desert environment. Identification of the key genes in the unsaturated fatty acid synthesis pathway could increase understanding of the biological regulatory mechanisms of fatty acid composition traits in plants and facilitate genetic manipulation of the

The kiwifruit cultivar Actinidia chinensis ‘Hort16A’ is resistant to the polyphagous armoured scale insect pest Hemiberlesia lataniae (Hemiptera: Diaspididae). A cDNA microarray consisting of 17,512 unigenes selected from over 132,000 expressed sequence tags (ESTs) was used to measure the transcriptomic profile of the A. chinensis ‘Hort16A’ canes in response to a controlled infestation of H. lataniae. After 2 days, 272 transcripts were differentially expressed. After 7 days, 5,284 (30%) transcripts were differentially expressed. The transcripts were grouped into 22 major functional categories using MapMan software. After 7 days, transcripts associated with photosynthesis (photosystem II) were significantly down-regulated, while those associated with secondary metabolism were significantly up-regulated. A total of 643 transcripts associated with response to stress were differentially expressed. This included biotic stress-related transcripts orthologous with pathogenesis related proteins, the phenylpropanoid pathway, NBS-LRR (R) genes, and receptor-like kinase–leucine rich repeat signalling proteins. While transcriptional studies are not conclusive in their own right, results were suggestive of a defence response involving both ETI and PTI, with predominance of the SA signalling pathway. Exogenous application of an SA-mimic decreased H. lataniae growth on A. chinensis ‘Hort16A’ plants in two laboratory experiments. PMID:26571404

Deep sequencing of transcriptomes has become an indispensable tool for biology, enabling expression levels for thousands of genes to be compared across multiple samples. Since transcript counts scale with sequencing depth, counts from different samples must be normalized to a common scale prior to comparison. We analyzed fifteen existing and novel algorithms for normalizing transcript counts, and evaluated the effectiveness of the resulting normalizations. For this purpose we defined two novel and mutually independent metrics: (1) the number of “uniform” genes (genes whose normalized expression levels have a sufficiently low coefficient of variation), and (2) low Spearman correlation between normalized expression profiles of gene pairs. We also define four novel algorithms, one of which explicitly maximizes the number of uniform genes, and compared the performance of all fifteen algorithms. The two most commonly used methods (scaling to a fixed total value, or equalizing the expression of certain ‘housekeeping’ genes) yielded particularly poor results, surpassed even by normalization based on randomly selected gene sets. Conversely, seven of the algorithms approached what appears to be optimal normalization. Three of these algorithms rely on the identification of “ubiquitous” genes: genes expressed in all the samples studied, but never at very high or very low levels. We demonstrate that these include a “core” of genes expressed in many tissues in a mutually consistent pattern, which is suitable for use as an internal normalization guide. The new methods yield robustly normalized expression values, which is a prerequisite for the identification of differentially expressed and tissue-specific genes as potential biomarkers. PMID:24223126

Faba bean is an important food crop worldwide. However, progress in faba bean genomics lags far behind that of model systems due to limited availability of genetic and genomic information. Using the Illumina platform the faba bean transcriptome from leaves of two lines (29H and Vf136) subjected to Ascochyta fabae infection have been characterized. De novo transcriptome assembly provided a total of 39,185 different transcripts that were functionally annotated, and among these, 13,266 were assigned to gene ontology against Arabidopsis. Quality of the assembly was validated by RT-qPCR amplification of selected transcripts differentially expressed. Comparison of faba bean transcripts with those of better-characterized plant genomes such as Arabidopsis thaliana, Medicago truncatula and Cicer arietinum revealed a sequence similarity of 68.3%, 72.8% and 81.27%, respectively. Moreover, 39,060 single nucleotide polymorphism (SNP) and 3,669 InDels were identified for genotyping applications. Mapping of the sequence reads generated onto the assembled transcripts showed that 393 and 457 transcripts were overexpressed in the resistant (29H) and susceptible genotype (Vf136), respectively. Transcripts involved in plant-pathogen interactions such as leucine rich proteins (LRR) or plant growth regulators involved in plant adaptation to abiotic and biotic stresses were found to be differently expressed in the resistant line. The results reported here represent the most comprehensive transcript database developed so far in faba bean, providing valuable information that could be used to gain insight into the pathways involved in the resistance mechanism against A. fabae and to identify potential resistance genes to be further used in marker assisted selection. PMID:26267359

Faba bean is an important food crop worldwide. However, progress in faba bean genomics lags far behind that of model systems due to limited availability of genetic and genomic information. Using the Illumina platform the faba bean transcriptome from leaves of two lines (29H and Vf136) subjected to Ascochyta fabae infection have been characterized. De novo transcriptome assembly provided a total of 39,185 different transcripts that were functionally annotated, and among these, 13,266 were assigned to gene ontology against Arabidopsis. Quality of the assembly was validated by RT-qPCR amplification of selected transcripts differentially expressed. Comparison of faba bean transcripts with those of better-characterized plant genomes such as Arabidopsis thaliana, Medicago truncatula and Cicer arietinum revealed a sequence similarity of 68.3%, 72.8% and 81.27%, respectively. Moreover, 39,060 single nucleotide polymorphism (SNP) and 3,669 InDels were identified for genotyping applications. Mapping of the sequence reads generated onto the assembled transcripts showed that 393 and 457 transcripts were overexpressed in the resistant (29H) and susceptible genotype (Vf136), respectively. Transcripts involved in plant-pathogen interactions such as leucine rich proteins (LRR) or plant growth regulators involved in plant adaptation to abiotic and biotic stresses were found to be differently expressed in the resistant line. The results reported here represent the most comprehensive transcript database developed so far in faba bean, providing valuable information that could be used to gain insight into the pathways involved in the resistance mechanism against A. fabae and to identify potential resistance genes to be further used in marker assisted selection. PMID:26267359

Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome. PMID:26241317

Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors‚ and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scaleanalysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system‚ which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scaletranscriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies. PMID:24226166

In order to understand the gene information, function, haloduric pathway (glycerolipid metabolism) and related key genes for Dunaliella viridis, we used Illumina HiSeqTM 2000 high-throughput sequencing technology to sequence its transcriptome. Trinity soft was used to assemble the data to form transcripts. Based on the Clusters of Orthologous Groups (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG ) databases, we carried out functional annotation and classification, pathway annotation, and the opening reading fragment (ORF) sequence prediction of transcripts. The key genes in the glycerolipid metabolism were analyzed. The results suggested that 81,593 transcripts were found, and 77,117 ORF sequences were predicted, accounting for 94.50% of all transcripts. COG classification results showed that 16,569 transcripts were assigned to 24 categories. GO classification annotated 76,436 transcripts. The number of transcripts for biologcial processes was 30,678, accounting for 40.14% of all transcripts. KEGG pathway analysis showed that 26,428 transcripts were annotated to 317 pathways, and 131 pathways were related to metabolism, accounting for 41.32% of all annotated pathways. Only one transcript was annotated as coding the key enzyme dihydroxyacetone kinase involved in the glycerolipid pathway. This enzyme could be related to glycerol biosynthesis under salt stress. This study further improved the gene information and laid the foundation of metabolic pathway research for Dunaliella viridis. PMID:26266786

Until recently, understanding the regulatory behavior of cells has been pursued through independent analysis of the transcriptome or the proteome. Based on the central dogma, it was generally assumed that there exist a direct correspondence between mRNA transcripts and generated protein expressions. However, recent studies have shown that the correlation between mRNA and Protein expressions can be low due to various factors such as different half lives and post transcription machinery. Thus, a joint analysis of the transcriptomic and proteomic data can provide useful insights that may not be deciphered from individual analysis of mRNA or protein expressions. This article reviews the existing major approaches for joint analysis of transcriptomic and proteomic data. We categorize the different approaches into eight main categories based on the initial algorithm and final analysis goal. We further present analogies with other domains and discuss the existing research problems in this area. PMID:24082820

Genomic studies of bacteria, archaea and viruses have provided insights into the microbial world by unveiling potential functional capabilities and molecular pathways. However, the rate of discovery has been slower among microbial eukaryotes, whose genomes are larger and more complex. Transcriptomic approaches provide a cost-effective alternative for examining genetic potential and physiological responses of microbial eukaryotes to environmental stimuli. In this study, we generated and compared the transcriptomes of four globally-distributed, bloom-forming prymnesiophyte algae: Prymnesium parvum, Chrysochromulina brevifilum, Chrysochromulina ericina and Phaeocystis antarctica. Our results revealed that the four transcriptomes possess a set of core genes that are similar in number and shared across all four organisms. The functional classifications of these core genes using the euKaryotic Orthologous Genes (KOG) database were also similar among the four study organisms. More broadly, when the frequencies of different cellular and physiological functions were compared with other protists, the species clustered by both phylogeny and nutritional modes. Thus, these clustering patterns provide insight into genomic factors relating to both evolutionary relationships as well as trophic ecology. This paper provides a novel comparative analysis of the transcriptomes of ecologically important and closely related prymnesiophyte protists and advances an emerging field of study that uses transcriptomics to reveal ecology and function in protists. PMID:24926657

Genomic studies of bacteria, archaea and viruses have provided insights into the microbial world by unveiling potential functional capabilities and molecular pathways. However, the rate of discovery has been slower among microbial eukaryotes, whose genomes are larger and more complex. Transcriptomic approaches provide a cost-effective alternative for examining genetic potential and physiological responses of microbial eukaryotes to environmental stimuli. In this study, we generated and compared the transcriptomes of four globally-distributed, bloom-forming prymnesiophyte algae: Prymnesium parvum, Chrysochromulina brevifilum, Chrysochromulina ericina and Phaeocystis antarctica. Our results revealed that the four transcriptomes possess a set of core genes that are similar in number and shared across all four organisms. The functional classifications of these core genes using the euKaryotic Orthologous Genes (KOG) database were also similar among the four study organisms. More broadly, when the frequencies of different cellular and physiological functions were compared with other protists, the species clustered by both phylogeny and nutritional modes. Thus, these clustering patterns provide insight into genomic factors relating to both evolutionary relationships as well as trophic ecology. This paper provides a novel comparative analysis of the transcriptomes of ecologically important and closely related prymnesiophyte protists and advances an emerging field of study that uses transcriptomics to reveal ecology and function in protists. PMID:24926657

The Chinese white wax scale insect, Ericerus pela, represents one of the most dramatic examples of sexual dimorphism in any insect species. In this study, we showed that although E. pela males display complete metamorphosis similar to holometabolous insects, the species forms the sister group to Acyrthosiphon pisum and cluster with hemimetabolous insects. The gene expression profile and Gene Ontology (GO) analyses revealed that the two sexes engaged in distinct developmental programs. In particular, female development appeared to prioritize the expression of genes related to cellular, metabolic, and developmental processes and to anatomical structure formation in nymphs. By contrast, male nymphal development is characterized by the significant down-regulation of genes involved in chitin, the respiratory system, and neurons. The wing and appendage morphogenesis, anatomical and tissue structure morphogenesis programs activated after male nymphal development. Transcription factors (that convey juvenile hormone or ecdysone signals, and Hox genes) and DNA methyltransferase were also differentially expressed between females and males. These results may indicate the roles that these differentially expressed genes play in regulating sexual dimorphism through orchestrating complex genetic programs. This differential expression was particularly prominent for processes linked to female development and wing development in males. PMID:25634031

The Chinese white wax scale insect, Ericerus pela, represents one of the most dramatic examples of sexual dimorphism in any insect species. In this study, we showed that although E. pela males display complete metamorphosis similar to holometabolous insects, the species forms the sister group to Acyrthosiphon pisum and cluster with hemimetabolous insects. The gene expression profile and Gene Ontology (GO) analyses revealed that the two sexes engaged in distinct developmental programs. In particular, female development appeared to prioritize the expression of genes related to cellular, metabolic, and developmental processes and to anatomical structure formation in nymphs. By contrast, male nymphal development is characterized by the significant down-regulation of genes involved in chitin, the respiratory system, and neurons. The wing and appendage morphogenesis, anatomical and tissue structure morphogenesis programs activated after male nymphal development. Transcription factors (that convey juvenile hormone or ecdysone signals, and Hox genes) and DNA methyltransferase were also differentially expressed between females and males. These results may indicate the roles that these differentially expressed genes play in regulating sexual dimorphism through orchestrating complex genetic programs. This differential expression was particularly prominent for processes linked to female development and wing development in males. PMID:25634031

Selecting colorectal cancer (CRC) patients likely to respond to therapy remains a clinical challenge. The objectives of this study were to establish which genes were differentially expressed with respect to treatment sensitivity and relate this to copy number in a panel of 15 CRC cell lines. Copy number variations of the identified genes were assessed in a cohort of CRCs. IC50’s were measured for 5-fluorouracil, oxaliplatin, and BEZ-235, a PI3K/mTOR inhibitor. Cell lines were profiled using array comparative genomic hybridisation, Illumina gene expression analysis, reverse phase protein arrays, and targeted sequencing of KRAS hotspot mutations. Frequent gains were observed at 2p, 3q, 5p, 7p, 7q, 8q, 12p, 13q, 14q, and 17q and losses at 2q, 3p, 5q, 8p, 9p, 9q, 14q, 18q, and 20p. Frequently gained regions contained EGFR, PIK3CA, MYC, SMO, TRIB1, FZD1, and BRCA2, while frequently lost regions contained FHIT and MACROD2. TRIB1 was selected for further study. Gene enrichment analysis showed that differentially expressed genes with respect to treatment response were involved in Wnt signalling, EGF receptor signalling, apoptosis, cell cycle, and angiogenesis. Stepwise integration of copy number and gene expression data yielded 47 candidate genes that were significantly correlated. PDCD6 was differentially expressed in all three treatment responses. Tissue microarrays were constructed for a cohort of 118 CRC patients and TRIB1 and MYC amplifications were measured using fluorescence in situ hybridisation. TRIB1 and MYC were amplified in 14.5% and 7.4% of the cohort, respectively, and these amplifications were significantly correlated (p≤0.0001). TRIB1 protein expression in the patient cohort was significantly correlated with pERK, Akt, and Caspase 3 expression. In conclusion, a set of candidate predictive biomarkers for 5-fluorouracil, oxaliplatin, and BEZ235 are described that warrant further study. Amplification of the putative oncogene TRIB1 has been described for

Background Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input. Results TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with

Background Capra hircus is an important economic livestock animal, and therefore, it is necessary to discover transcriptome information about their reproductive performance. In this study, we performed de novo transcriptome sequencing to produce the first transcriptome dataset for the goat ovary using high-throughput sequencing technologies. The result will contribute to research on goat reproductive performance. Method and Results RNA-seq analysis generated more than 38.8 million clean paired end (PE) reads, which were assembled into 80,069 unigenes (mean size = 619 bp). Based on sequence similarity searches, 64,824 (60.6%) genes were identified, among which 29,444 and 11,271 unigenes were assigned to Gene Ontology (GO) categories and Clusters of Orthologous Groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG) showed that 27,766 (63.4%) unigenes were mapped to 258 KEGG pathways. Furthermore, we investigated the transcriptome differences of goat ovaries at two different ages using a tag-based digital gene expression system. We obtained a sequencing depth of over 5.6 million and 5.8 million tags for the two ages and identified a large number of genes associated with reproductive hormones, ovulatory cycle and follicle. Moreover, many antisense transcripts and novel transcripts were found; clusters with similar differential expression patterns, enriched GO terms and metabolic pathways were revealed for the first time with regard to the differentially expressed genes. Conclusions The transcriptome provides invaluable new data for a functional genomic resource and future biological research in Capra hircus, and it is essential for the in-depth study of candidate genes in breeding programs. PMID:25822507

Transcriptomics meta-analysis aims at re-using existing data to derive novel biological hypotheses, and is motivated by the public availability of a large number of independent studies. Current methods are based on breaking down studies into multiple comparisons between phenotypes (e.g. disease vs. healthy), based on the studies' experimental designs, followed by computing the overlap between the resulting differential expression signatures. While useful, in this methodology each study yields multiple independent phenotype comparisons, and connections are established not between studies, but rather between subsets of the studies corresponding to phenotype comparisons. We propose a rank-based statistical meta-analysis framework that establishes global connections between transcriptomics studies without breaking down studies into sets of phenotype comparisons. By using a rank product method, our framework extracts global features from each study, corresponding to genes that are consistently among the most expressed or differentially expressed genes in that study. Those features are then statistically modelled via a term-frequency inverse-document frequency (TF-IDF) model, which is then used for connecting studies. Our framework is fast and parameter-free; when applied to large collections of Homo sapiens and Streptococcus pneumoniae transcriptomics studies, it performs better than similarity-based approaches in retrieving related studies, using a Medical Subject Headings gold standard. Finally, we highlight via case studies how the framework can be used to derive novel biological hypotheses regarding related studies and the genes that drive those connections. Our proposed statistical framework shows that it is possible to perform a meta-analysis of transcriptomics studies with arbitrary experimental designs by deriving global expression features rather than decomposing studies into multiple phenotype comparisons. PMID:24586684

Even with recent reductions in sequencing costs, most plants lack the genomic resources required for successful short-read transcriptome analyses as performed routinely in model species. Several approaches for the analysis of short-read transcriptome data are reviewed for nonmodel species for which the genome of a close relative is used as the reference genome. Two approaches using a data set from Phytophthora-challenged Rubus idaeus (red raspberry) are compared. Over 70000000 86-nt Illumina reads derived from R. idaeus roots were aligned to the Fragaria vesca genome using publicly available informatics tools (Bowtie/TopHat and Cufflinks). Alignment identified 16956 putatively expressed genes. De novo assembly was performed with the same data set and a publicly available transcriptome assembler (Trinity). A BLAST search with a maximum e-value threshold of 1.0 × 10(-3) revealed that over 36000 transcripts had matches to plants and over 500 to Phytophthora. Gene expression estimates from alignment to F. vesca and de novo assembly were compared for raspberry (Pearson's correlation = 0.730). Together, alignment to the genome of a close relative and de novo assembly constitute a powerful method of transcriptomeanalysis in nonmodel organisms. Alignment to the genome of a close relative provides a framework for differential expression testing if alignments are made to the predefined gene-space of a close relative and de novo assembly provides a more robust method of identifying unique sequences and sequences from other organisms in a system. These methods are considered experimental in nonmodel systems, but can be used to generate resources and specific testable hypotheses. PMID:22301897

Background Maize is one of the most important crops in the world. With the exponentially increasing population and the need for ever increased food and feed production, an increased yield of maize grain (as well as rice, wheat and other grains) will be critical. Maize grain development is understood from the perspective of morphology, hormone responses, and storage reserve accumulation. This includes various studies on gene expression during embryo development and maturation but a global study of gene expression of the embryo has not been possible until recently. Transcriptomeanalysis is a powerful new tool that can be used to understand the genetic basis of embryo maturation. Results We undertook a transcriptomicanalysis of normal maturing embryos at 15, 21 and 27 days after pollination (DAP), of one elite maize germplasm line that was utilized in crosses to transgenic plants. More than 19,000 genes were analyzed by this method and the challenge was to select subsets of genes that are vitally important to embryo development and maturation for the initial analysis. We describe the changes in expression for genes relating to primary metabolic pathways, DNA synthesis, late embryogenesis proteins and embryo storage proteins, shown through transcriptomeanalysis and confirmed levels of transcription for some genes in the transcriptome using qRT-PCR. Conclusions Numerous genes involved in embryo maturation have been identified, many of which show changes in expression level during the progression from 15 to 27 DAP. An expected array of genes involved in primary metabolism was identified. Moreover, more than 30% of transcripts represented un-annotated genes, leaving many functions to be discovered. Of particular interest are the storage protein genes, globulin-1, globulin-2 and an unidentified cupin family gene. When expressing foreign proteins in maize, the globulin-1 promoter is most often used, but this cupin family gene has much higher expression and may be a

Sika deer is of great commercial value because their antlers are used in tonics and alternative medicine and their meat is healthy and delicious. The goal of this study was to generate transcript sequences from sika deer for functional genomic analyses and to identify the transcripts that demonstrate tissue-specific, age-dependent differential expression patterns. These sequences could enhance our understanding of the molecular mechanisms underlying sika deer growth and development. In the present study, we performed de novo transcriptome assembly and profiling analysis across ten tissue types and four developmental stages (juvenile, adolescent, adult, and aged) of sika deer, using Illumina paired-end tag (PET) sequencing technology. A total of 1,752,253 contigs with an average length of 799 bp were generated, from which 1,348,618 unigenes with an average length of 590 bp were defined. Approximately 33.2 % of these (447,931 unigenes) were then annotated in public protein databases. Many sika deer tissue-specific, age-dependent unigenes were identified. The testes have the largest number of tissue-enriched unigenes, and some of them were prone to develop new functions for other tissues. Additionally, our transcriptome revealed that the juvenile-adolescent transition was the most complex and important stage of the sika deer life cycle. The present work represents the first multiple tissue transcriptomeanalysis of sika deer across four developmental stages. The generated data not only provide a functional genomics resource for future biological research on sika deer but also guide the selection and manipulation of genes controlling growth and development. PMID:27423230

Transcriptomeanalysis has been a key area of biological inquiry for decades. Over the years, research in the field has progressed from candidate gene-based detection of RNAs using Northern blotting to high-throughput expression profiling driven by the advent of microarrays. Next-generation sequencing technologies have revolutionized transcriptomics by providing opportunities for multidimensional examinations of cellular transcriptomes in which high-throughput expression data are obtained at a single-base resolution. PMID:19715439

Ginkgo biloba is a dioecious species native to China with medicinally and phylogenetically important characteristics; however, genomic resources for this species are limited. In this study, we performed the first transcriptome sequencing for Ginkgo kernels at five time points using Illumina paired-end sequencing. Approximately 25.08-Gb clean reads were obtained, and 68,547 unigenes with an average length of 870 bp were generated by de novo assembly. Of these unigenes, 29,987 (43.74%) were annotated in publicly available plant protein database. A total of 3,869 genes were identified as significantly differentially expressed, and enrichment analysis was conducted at different time points. Furthermore, metabolic pathway analysis revealed that 66 unigenes were responsible for terpenoid backbone biosynthesis, with up to 12 up-regulated unigenes involved in the biosynthesis of ginkgolide and bilobalide. Differential gene expression analysis together with real-time PCR experiments indicated that the synthesis of bilobalide may have interfered with the ginkgolide synthesis process in the kernel. These data can remarkably expand the existing transcriptome resources of Ginkgo, and provide a valuable platform to reveal more on developmental and metabolic mechanisms of this species. PMID:26500663

The fast development of next-generation sequencing technology presents a major computational challenge for data processing and analysis. A fast algorithm, de Bruijn graph has been successfully used for genome DNA de novo assembly; nevertheless, its performance for transcriptome assembly is unclear. In this study, we used both simulated and real RNA-Seq data, from either artificial RNA templates or human transcripts, to evaluate five de novo assemblers, ABySS, Mira, Trinity, Velvet and Oases. Of these assemblers, ABySS, Trinity, Velvet and Oases are all based on de Bruijn graph, and Mira uses an overlap graph algorithm. Various numbers of RNA short reads were selected from the External RNA Control Consortium (ERCC) data and human chromosome 22. A number of statistics were then calculated for the resulting contigs from each assembler. Each experiment was repeated multiple times to obtain the mean statistics and standard error estimate. Trinity had relative good performance for both ERCC and human data, but it may not consistently generate full length transcripts. ABySS was the fastest method but its assembly quality was low. Mira gave a good rate for mapping its contigs onto human chromosome 22, but its computational speed is not satisfactory. Our results suggest that transcript assembly remains a challenge problem for bioinformatics society. Therefore, a novel assembler is in need for assembling transcriptome data generated by next generation sequencing technique. PMID:23393031

Monitoring global gene expression provides insight into how genes and regulatory signals work together to guide embryo development. The fields of developmental biology and teratology are now confronted with the need for automated access to a reference library of gene-expression signatures that benchmark programmed (genetic) and adaptive (environmental) regulation of the embryonic transcriptome. Such a library must be constructed from highly-distributed microarray data. Birth Defects Systems Manager (BDSM), an open access knowledge management system, provides custom software to mine public microarray data focused on developmental health and disease. The present study describes tools for seamless data integration in the BDSM library (MetaSample, MetaChip, CIAeasy) using the QueryBDSM module. A field test of the prototype was run using published microarray data series derived from a variety of laboratories, experiments, microarray platforms, organ systems, and developmental stages. The datasets focused on several developing systems in the mouse embryo, including preimplantation stages, heart and nerve development, testis and ovary development, and craniofacial development. Using BDSM data integration tools, a gene-expression signature for 346 genes was resolved that accurately classified samples by organ system and developmental sequence. The module builds a potential for the BDSM approach to decipher a large number developmental processes through comparative bioinformatics analysis of embryological systems at-risk for specific defects, using multiple scenarios to define the range of probabilities leading from molecular phenotype to clinical phenotype. We conclude that an integrative analysis of global gene-expression of the developing embryo can form the foundation for constructing a reference library of signaling pathways and networks for normal and abnormal regulation of the embryonic transcriptome. These tools are available free of charge from the web-site http

Monitoring global gene expression provides insight into how genes and regulatory signals work together to guide embryo development. The fields of developmental biology and teratology are now confronted with the need for automated access to a reference library of gene-expression signatures that benchmark programmed (genetic) and adaptive (environmental) regulation of the embryonic transcriptome. Such a library must be constructed from highly-distributed microarray data. Birth Defects Systems Manager (BDSM), an open access knowledge management system, provides custom software to mine public microarray data focused on developmental health and disease. The present study describes tools for seamless data integration in the BDSM library (MetaSample, MetaChip, CIAeasy) using the QueryBDSM module. A field test of the prototype was run using published microarray data series derived from a variety of laboratories, experiments, microarray platforms, organ systems, and developmental stages. The datasets focused on several developing systems in the mouse embryo, including preimplantation stages, heart and nerve development, testis and ovary development, and craniofacial development. Using BDSM data integration tools, a gene-expression signature for 346 genes was resolved that accurately classified samples by organ system and developmental sequence. The module builds a potential for the BDSM approach to decipher a large number developmental processes through comparative bioinformatics analysis of embryological systems at-risk for specific defects, using multiple scenarios to define the range of probabilities leading from molecular phenotype to clinical phenotype. We conclude that an integrative analysis of global gene-expression of the developing embryo can form the foundation for constructing a reference library of signaling pathways and networks for normal and abnormal regulation of the embryonic transcriptome. These tools are available free of charge from the web-site http

High–throughput technologies used to interrogate transcriptomes have been generating a great amount of publicly available gene expression data. For raw diseases that lack of clinical samples and research funding, there is a practical benefit to jointly analyze existing datasets commonly related to a specific rare disease. In this study, we collected a number of independently generated transcriptome data sets from four species: Human, Fly, Mouse and Worm. All data sets included samples with both normal and abnormal mitochondrial functions. We reprocessed each data set to standardize format, scale and gene annotation and used HomoloGene database to map genes between species. Standard procedure was also applied to compare gene expression profiles of normal and abnormal mitochondrial functions to identify differentially expressed genes. We further used meta–analysis and other integrative analyses to recognize patterns across data sets and species. Novel insights related to mitochondrial dysfunctions was revealed via these analyses, such as a group of genes consistently dysregulated by impaired mitochondrial function in multiple species. This study created a template for the study of rare diseases using genomic technologies and advanced statistical methods. All data and results generated by this study are freely available and stored at http://goo.gl/nOGWC2, to support further data mining. PMID:24981076

Background Panax ginseng Meyer is a traditional medicinal plant famous for its strong therapeutic effects and serves as an important herbal medicine. To understand and manipulate genes involved in secondary metabolic pathways including ginsenosides, transcriptome profiling of P. ginseng is essential. Methods RNA-seq analysis of adventitious roots of two P. ginseng cultivars, Chunpoong (CP) and Cheongsun (CS), was performed using the Illumina HiSeq platform. After transcripts were assembled, expression profiling was performed. Results Assemblies were generated from ∼85 million and ∼77 million high-quality reads from CP and CS cultivars, respectively. A total of 35,527 and 27,716 transcripts were obtained from the CP and CS assemblies, respectively. Annotation of the transcriptomes showed that approximately 90% of the transcripts had significant matches in public databases. We identified several candidate genes involved in ginsenoside biosynthesis. In addition, a large number of transcripts (17%) with different gene ontology designations were uniquely detected in adventitious roots compared to normal ginseng roots. Conclusion This study will provide a comprehensive insight into the transcriptome of ginseng adventitious roots, and a way for successful transcriptomeanalysis and profiling of resource plants with less genomic information. The transcriptome profiling data generated in this study are available in our newly created adventitious root transcriptome database (http://im-crop.snu.ac.kr/transdb/index.php) for public use. PMID:25379008

Purpose Dry eye disease is a common condition associated with age-related meibomian gland dysfunction (ARMGD). We have previously shown that ARMGD occurs in old mice, similar to that observed in human patients with MGD. To begin to understand the mechanism underlying ARMGD, we generated transcriptome profiles of eyelids excised from young and old mice of both sexes. Methods Male and female C57BL/6 mice were euthanized at ages of 3 months or 2 years and their lower eyelids removed, the conjunctival epithelium scrapped off, and the tarsal plate, containing the meibomian glands, dissected from the overlying muscle and lid epidermis. RNA was isolated, enriched, and transcribed into cDNA and processed to generate four non-stranded libraries with distinct bar codes on each adaptor. The libraries were then sequenced and mapped to the mm10 reference genome, and expression results were gathered as reads per length of transcript in kilobases per million mapped reads (RPKM) values. Differential gene expression analyses were performed using CyberT. Results Approximately 55 million reads were generated from each library. Expression data indicated that about 15,000 genes were expressed in these tissues. Of the genes that showed more than twofold significant differences in either young or old tissue, 698 were identified as differentially expressed. According to the Gene Ontology (GO) analysis, the cellular, developmental, and metabolic processes were found to be highly represented with Wnt function noted to be altered in the aging mouse. Conclusions The RNA sequencing data identified several signaling pathways, including fibroblast growth factor (FGF) and Wnt that were altered in the meibomian glands of aging mice. PMID:27279727

Background The plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of (1 → 3)-β-linked glucose with a (1 → 6)-β-glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan production, has not been elucidated yet. In order to provide a basis for genetic and metabolic engineering approaches, we studied scleroglucan and oxalate biosynthesis in S. rolfsii using different transcriptomic approaches. Results Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding ~350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified ~800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. From these, candidate genes were identified which could represent potential leads for targeted modification of the S. rolfsii metabolism for increased scleroglucan yields. Conclusions The results presented in this paper provide for the first time genomic and transcriptomic data about S. rolfsii and demonstrate the power and usefulness of combined transcriptome sequencing and comparative microarray analysis. The data obtained allowed us to predict the biosynthetic pathways of scleroglucan and oxalate synthesis and to

Citrullus colocynthis is a very drought tolerant species, closely related to watermelon (C. lanatus var. lanatus), an economically important cucurbit crop. Drought is a threat to plant growth and development, and the discovery of drought inducible genes with various functions is of great importance. We used high throughput mRNA Illumina sequencing technology and bioinformatic strategies to analyze the C. colocynthis leaf transcriptome under drought treatment. Leaf samples at four different time points (0, 24, 36, or 48 hours of withholding water) were used for RNA extraction and Illumina sequencing. qRT-PCR of several drought responsive genes was performed to confirm the accuracy of RNA sequencing. Leaf transcriptomeanalysis provided the first glimpse of the drought responsive transcriptome of this unique cucurbit species. A total of 5038 full-length cDNAs were detected, with 2545 genes showing significant changes during drought stress. Principle component analysis indicated that drought was the major contributing factor regulating transcriptome changes. Up regulation of many transcription factors, stress signaling factors, detoxification genes, and genes involved in phytohormone signaling and citrulline metabolism occurred under the water deficit conditions. The C. colocynthis transcriptome data highlight the activation of a large set of drought related genes in this species, thus providing a valuable resource for future functional analysis of candidate genes in defense of drought stress. PMID:25118696

Sequencing of the Populus trichocarpa genome creates an opportunity to describe the transcriptome of a woody perennial species and establish an atlas of gene expression. A comparison with the transcriptomes of other species can also define genes that are conserved or diverging in plant species. Here, the transcriptome in vegetative organs of the P. trichocarpa reference genotype Nisqually-1 was characterized. A comparison with Arabidopsis thaliana orthologs was used to distinguish gene functional categories that may be evolving differently in a woody perennial and an annual herbaceous species. A core set of genes expressed in common among vegetative organs was detected, as well as organ-specific genes. Statistical tests identified chromatin domains, where adjacent genes were expressed more frequently than expected by chance. Extensive divergence was detected in the expression patterns of A. thaliana and P. trichocarpa orthologs, but transcription of a small number of genes appeared to have remained conserved in the two species. Despite separation of lineages for over 100 million yr, these results suggest that selection has limited transcriptional divergence of genes associated with some essential functions in A. thaliana and P. trichocarpa. However, extensive remodeling of transcriptional networks indicates that expression regulation may be a key determinant of plant diversity.

In this paper we present a de novo assembly of the transcriptome of the damselfly, Enallagma hageni, through the use of 454 pyrosequencing. E. hageni is a member of the suborder Zygoptera within the order Odonata, and the Odonata are the basal lineage of the winged insects (Pterygota). To date, sequence data used in phylogenetic analysis of Enallagma species have been derived from either mtDNA or ribosomal nuclear DNA. This transcriptome contained 31,661 contigs that were assembled and translated into 14,813 individual open reading frames. Using these data, we constructed an extensive dataset of 634 orthologous nuclear protein-coding genes across 11 species of Arthropoda, and used Bayesian techniques to elucidate Enallagma's place in the Arthropod phylogenetic tree. Additionally, we demonstrate that the Enallagma transcriptome contains 169 genes that are evolving at rates that differ relative to the rest of the transcriptome (29 accelerated and 140 decreased), and through multiple Gene Ontology searches and clustering methods, we present the first functional-annotation of any palaeopteran's transcriptome in the literature. PMID:23550132

The transcriptome of Thinopyrum elongatum under water deficit stress was analyzed using RNA-Seq technology. The results showed that genes involved in processes of amplification of stress signaling, reductions in oxidative damage, creation of protectants, and roots development were expressed differently, which played an important role in the response to water deficit. The Th. elongatum transcriptome research highlights the activation of a large set of water deficit-related genes in this species and provides a valuable resource for future functional analysis of candidate genes in the water deficit stress response. PMID:25722968

Plant breeders have focused much attention on polyploid trees because of their importance to forestry. To evaluate the impact of intraspecies genome duplication on the transcriptome, a series of Betula platyphylla autotetraploids and diploids were generated from four full-sib families. The phenotypes and transcriptomes of these autotetraploid individuals were compared with those of diploid trees. Autotetraploids were generally superior in breast-height diameter, volume, leaf, fruit and stoma and were generally inferior in height compared to diploids. Transcriptome data revealed numerous changes in gene expression attributable to autotetraploidization, which resulted in the upregulation of 7052 unigenes and the downregulation of 3658 unigenes. Pathway analysis revealed that the biosynthesis and signal transduction of indoleacetate (IAA) and ethylene were altered after genome duplication, which may have contributed to phenotypic changes. These results shed light on variations in birch autotetraploidization and help identify important genes for the genetic engineering of birch trees. PMID:23202935

The non-human primate reference transcriptome resource (NHPRTR, available online at http://nhprtr.org/) aims to generate comprehensive RNA-seq data from a wide variety of non-human primates (NHPs), from lemurs to hominids. In the 2012 Phase I of the NHPRTR project, 19 billion fragments or 3.8 terabases of transcriptome sequences were collected from pools of ∼20 tissues in 15 species and subspecies. Here we describe a major expansion of NHPRTR by adding 10.1 billion fragments of tissue-specific RNA-seq data. For this effort, we selected 11 of the original 15 NHP species and subspecies and constructed total RNA libraries for the same ∼15 tissues in each. The sequence quality is such that 88% of the reads align to human reference sequences, allowing us to compute the full list of expression abundance across all tissues for each species, using the reads mapped to human genes. This update also includes improved transcript annotations derived from RNA-seq data for rhesus and cynomolgus macaques, two of the most commonly used NHP models and additional RNA-seq data compiled from related projects. Together, these comprehensive reference transcriptomes from multiple primates serve as a valuable community resource for genome annotation, gene dynamics and comparative functional analysis. PMID:25392405

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora. PMID:26690126

Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora. PMID:26690126

The transcriptome of a lager brewing yeast (Saccharomyces carlsbergensis, syn. of S. pastorianus), was analysed at 12 different time points spanning a production-scale lager beer fermentation. Generally, the average expression rapidly increased and had a maximum value on day 2, then decreased as the sugar got consumed. Especially genes involved in protein and lipid biosynthesis or glycolysis were highly expressed during the beginning of the fermentation. Similarities as well as significant differences in expression profiles could be observed when comparing to a previous transcriptomeanalysis of a laboratory yeast grown in YPD. The regional distribution of various expression levels on the chromosomes appeared to be random or near-random and no reduction in expression near telomeres was observed. PMID:12702272

Sarracenia species (pitcher plants) are carnivorous plants which obtain a portion of their nutrients from insects captured in the pitchers. To investigate these plants, we sequenced the transcriptome of two species, Sarracenia psittacina and Sarracenia purpurea, using Roche 454 pyrosequencing technology. We obtained 46 275 and 36 681 contigs by de novo assembly methods for S. psittacina and S. purpurea, respectively, and further identified 16 163 orthologous contigs between them. Estimation of synonymous substitution rates between orthologous and paralogous contigs indicates the events of genome duplication and speciation within the Sarracenia genus both occurred ∼2 million years ago. The ratios of synonymous and non-synonymous substitution rates indicated that 491 contigs have been under positive selection (K(a)/K(s) > 1). Significant proportions of these contigs were involved in functions related to binding activity. We also found that the greatest sequence similarity for both of these species was to Vitis vinifera, which is most consistent with a non-current classification of the order Ericales as an asterid. This study has provided new insights into pitcher plants and will contribute greatly to future research on this genus and its distinctive ecological adaptations. PMID:21676972

Dormancy transitions in crown buds of leafy spurge were investigated using 23K element cDNA microarrays. These data represent the first large-scaletranscriptomeanalysis of dormancy in underground buds of an herbaceous perennial species. Crown buds collected monthly from August through December, ov...

Saliva is known to play a crucial role in insect feeding behavior and virus transmission. Currently, little is known about the salivary glands and saliva of thrips, despite the fact that Frankliniella occidentalis (Pergande) (the western flower thrips) is a serious pest due to its destructive feeding, wide host range, and transmission of tospoviruses. As a first step towards characterizing thrips salivary gland functions, we sequenced the transcriptome of the primary salivary glands of F. occidentalis using short read sequencing (Illumina) technology. A de novo-assembled transcriptome revealed 31,392 high quality contigs with an average size of 605 bp. A total of 12,166 contigs had significant BLASTx or tBLASTx hits (E≤1.0E-6) to known proteins, whereas a high percentage (61.24%) of contigs had no apparent protein or nucleotide hits. Comparison of the F. occidentalis salivary gland transcriptome (sialotranscriptome) against a published F. occidentalis full body transcriptome assembled from Roche-454 reads revealed several contigs with putative annotations associated with salivary gland functions. KEGG pathway analysis of the sialotranscriptome revealed that the majority (18 out of the top 20 predicted KEGG pathways) of the salivary gland contig sequences match proteins involved in metabolism. We identified several genes likely to be involved in detoxification and inhibition of plant defense responses including aldehyde dehydrogenase, metalloprotease, glucose oxidase, glucose dehydrogenase, and regucalcin. We also identified several genes that may play a role in the extra-oral digestion of plant structural tissues including β-glucosidase and pectin lyase; and the extra-oral digestion of sugars, including α-amylase, maltase, sucrase, and α-glucosidase. This is the first analysis of a sialotranscriptome for any Thysanopteran species and it provides a foundational tool to further our understanding of how thrips interact with their plant hosts and the viruses they

Saliva is known to play a crucial role in insect feeding behavior and virus transmission. Currently, little is known about the salivary glands and saliva of thrips, despite the fact that Frankliniella occidentalis (Pergande) (the western flower thrips) is a serious pest due to its destructive feeding, wide host range, and transmission of tospoviruses. As a first step towards characterizing thrips salivary gland functions, we sequenced the transcriptome of the primary salivary glands of F. occidentalis using short read sequencing (Illumina) technology. A de novo-assembled transcriptome revealed 31,392 high quality contigs with an average size of 605 bp. A total of 12,166 contigs had significant BLASTx or tBLASTx hits (E≤1.0E−6) to known proteins, whereas a high percentage (61.24%) of contigs had no apparent protein or nucleotide hits. Comparison of the F. occidentalis salivary gland transcriptome (sialotranscriptome) against a published F. occidentalis full body transcriptome assembled from Roche-454 reads revealed several contigs with putative annotations associated with salivary gland functions. KEGG pathway analysis of the sialotranscriptome revealed that the majority (18 out of the top 20 predicted KEGG pathways) of the salivary gland contig sequences match proteins involved in metabolism. We identified several genes likely to be involved in detoxification and inhibition of plant defense responses including aldehyde dehydrogenase, metalloprotease, glucose oxidase, glucose dehydrogenase, and regucalcin. We also identified several genes that may play a role in the extra-oral digestion of plant structural tissues including β-glucosidase and pectin lyase; and the extra-oral digestion of sugars, including α-amylase, maltase, sucrase, and α-glucosidase. This is the first analysis of a sialotranscriptome for any Thysanopteran species and it provides a foundational tool to further our understanding of how thrips interact with their plant hosts and the viruses

Background Some species of the whitefly Bemisia tabaci complex cause tremendous losses to crops worldwide through feeding directly and virus transmission indirectly. The primary salivary glands of whiteflies are critical for their feeding and virus transmission. However, partly due to their tiny size, research on whitefly salivary glands is limited and our knowledge on these glands is scarce. Methodology/Principal Findings We sequenced the transcriptome of the primary salivary glands of the Mediterranean species of B. tabaci complex using an effective cDNA amplification method in combination with short read sequencing (Illumina). In a single run, we obtained 13,615 unigenes. The quantity of the unigenes obtained from the salivary glands of the whitefly is at least four folds of the salivary gland genes from other plant-sucking insects. To reveal the functions of the primary glands, sequence similarity search and comparisons with the whole transcriptome of the whitefly were performed. The results demonstrated that the genes related to metabolism and transport were significantly enriched in the primary salivary glands. Furthermore, we found that a number of highly expressed genes in the salivary glands might be involved in secretory protein processing, secretion and virus transmission. To identify potential proteins of whitefly saliva, the translated unigenes were put into secretory protein prediction. Finally, 295 genes were predicted to encode secretory proteins and some of them might play important roles in whitefly feeding. Conclusions/Significance: The combined method of cDNA amplification, Illumina sequencing and de novo assembly is suitable for transcriptomicanalysis of tiny organs in insects. Through analysis of the transcriptome, genomic features of the primary salivary glands were dissected and biologically important proteins, especially secreted proteins, were predicted. Our findings provide substantial sequence information for the primary salivary glands

Background The Eastern hive honey bee, Apis cerana cerana is a native and widely bred honey bee species in China. Molecular biology research about this honey bee species is scarce, and genomic information for A. c. cerana is not currently available. Transcriptome and expression profiling data for this species are therefore important resources needed to better understand the biological mechanisms of A. c. cerana. In this study, we obtained the transcriptome information of A. c. cerana by RNA-sequencing and compared gene expression differences between queens and workers of A. c. cerana by digital gene expression (DGE) analysis. Results Using high-throughput Illumina RNA sequencing we obtained 51,581,510 clean reads corresponding to 4.64 Gb total nucleotides from a single run. These reads were assembled into 46,999 unigenes with a mean length of 676 bp. Based on a sequence similarity search against the five public databases (NR, Swissport, GO, COG, KEGG) with a cut-off E-value of 10−5 using BLASTX, a total of 24,630 unigenes were annotated with gene descriptions, gene ontology terms, or metabolic pathways. Using these transcriptome data as references we analyzed the gene expression differences between the queens and workers of A. c. cerana using a tag-based digital gene expression method. We obtained 5.96 and 5.66 million clean tags from the queen and worker samples, respectively. A total of 414 genes were differentially expressed between them, with 189 up-regulated and 225 down-regulated in queens. Conclusions Our transcriptome data provide a comprehensive sequence resource for future A. c. cerana study, establishing an important public information platform for functional genomic studies in A. c. cerana. Furthermore, the DGE data provide comprehensive gene expression information for the queens and workers, which will facilitate our understanding of the molecular mechanisms of the different physiological aspects of the two castes. PMID:23112877

The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scaletranscriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals. PMID:25072156

The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scaletranscriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals. PMID:25072156

Wintersweet (Chimonanthus praecox) is familiar as a garden plant and woody ornamental flower. On account of its unique flowering time and strong fragrance, it has a high ornamental and economic value. Despite a long history of human cultivation, our understanding of wintersweet genetics and molecular biology remains scant, reflecting a lack of basic genomic and transcriptomic data. In this study, we assembled three cDNA libraries, from three successive stages in flower development, designated as the flower bud with displayed petal, open flower and senescing flower stages. Using the Illumina RNA-Seq method, we obtained 21,412,928, 26,950,404, 24,912,954 qualified Illumina reads, respectively, for the three successive stages. The pooled reads from all three libraries were then assembled into 106,995 transcripts, 51,793 of which were annotated in the NCBI non-redundant protein database. Of these annotated sequences, 32,649 and 21,893 transcripts were assigned to gene ontology categories and clusters of orthologous groups, respectively. We could map 15,587 transcripts onto 312 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at the open flower and senescing flower stages. An analysis of differentially expressed genes involved in plant hormone signal transduction pathways indicated that although flower opening and senescence may be independent of the ethylene signaling pathway in wintersweet, salicylic acid may be involved in the regulation of flower senescence. We also succeeded in isolating key genes of floral scent biosynthesis and proposed a biosynthetic pathway for monoterpenes and sesquiterpenes in wintersweet flowers, based on the annotated sequences. This comprehensive transcriptomicanalysis presents fundamental information on the genes and pathways which are involved in flower development in wintersweet. And our data

Recent advances in high-throughput technologies enable quantitative monitoring of the abundance of various biological molecules and allow determination of their variation between biological states on a genomic scale. Two popular platforms areDNA microarrays to measure messenger RNA transcript levels, and gel-free proteomic analyses to determine protein abundance. Obviously, no single approach can fully unravel the complexities of fundamental biology and it is equally clear that integrative analysis of multiple levels of gene expression would be valuable in this endeavor. However, most integrative transcriptomic and proteomic studies have thus far either failed to find a correlation or have only observed a weak correlation. It is evident that this failure is not biologically based, but rather is related the inadequacy of available statistical tools to compensate for biases in the data collection methodologies. To address this issue, attempts have recently been made to systematically investigate the correlation patterns between transcriptomic and proteomic datasets, and to develop more sophisticated statistical tools to improve the chances of capturing a relationship. The goal of these investigations is to enhance our understanding of the relationship between transcriptome and proteome data so that integrative analyses may be utilized to reveal new biological insights that are not accessible through one dimensional datasets. In this review, we outline some of the challenges associated with integrative analyses and present some preliminary solutions based on progress being made in recent years. In addition, some new applications of integrated transcriptomic and proteomic analysis to the investigation of post-transcriptional regulation will also be discussed.

Gene-expression heterogeneity among individual cells determines the fate of a bacterial population. Here we report the first bacterial single-cell RNA sequencing (RNA-seq), BaSiC RNA-seq, a method integrating RNA isolation, cDNA synthesis and amplification, and RNA-seq analysis of the whole transcriptome of single cyanobacterium Synechocystis sp. PCC 6803 cells which typically contain approximately 5-7 femtogram total RNA per cell. We applied the method to 3 Synechocystis single cells at 24 h and 3 single cells at 72 h after nitrogen-starvation stress treatment, as well as their bulk-cell controls under the same conditions, to determine the heterogeneity upon environmental stress. With 82-98% and 31-48% of all putative Synechocystis genes identified in single cells of 24 and 72 h, respectively, the results demonstrated that the method could achieve good identification of the transcripts in single bacterial cells. In addition, the preliminary results from nitrogen-starved cells also showed a possible increasing gene-expression heterogeneity from 24 h to 72 h after nitrogen starvation stress. Moreover, preliminary analysis of single-cell transcriptomic datasets revealed that genes from the "Mobile elements" functional category have the most significant increase of gene-expression heterogeneity upon stress, which was further confirmed by single-cell RT-qPCR analysis of gene expression in 24 randomly selected cells. PMID:26331465

RNA-seq has emerged as the technology of choice to quantify gene expression. This technology is a convenient accurate tool to quantify diurnal changes in gene expression, gene discovery, differential use of promoters, and splice variants for all genes expressed in a single tissue. Thus, RNA-seq experiments provide sequence information and absolute expression values about transcripts in addition to relative quantification available with microarrays or qRT-PCR. The depth of information by sequencing requires careful assessment of RNA intactness and DNA contamination. Although the RNA-seq is comparatively recent, a standard analysis framework has emerged with the packages of Bowtie2, TopHat, and Cufflinks. With rising popularity of RNA-seq tools have become manageable for researchers without much bioinformatical knowledge or programming skills. Here, we present a workflow for a RNA-seq experiment from experimental planning to biological data extraction. PMID:24792045

OBJECTIVE Diabetic kidney disease (DKD) is the single leading cause of kidney failure in the U.S., for which a cure has not yet been found. The aim of our study was to provide an unbiased catalog of gene-expression changes in human diabetic kidney biopsy samples. RESEARCH DESIGN AND METHODS Affymetrix expression arrays were used to identify differentially regulated transcripts in 44 microdissected human kidney samples. DKD samples were significant for their racial diversity and decreased glomerular filtration rate (~25–35 mL/min). Stringent statistical analysis, using the Benjamini-Hochberg corrected two-tailed t test, was used to identify differentially expressed transcripts in control and diseased glomeruli and tubuli. Two different web-based algorithms were used to define differentially regulated pathways. RESULTS We identified 1,700 differentially expressed probesets in DKD glomeruli and 1,831 in diabetic tubuli, and 330 probesets were commonly differentially expressed in both compartments. Pathway analysis highlighted the regulation of Ras homolog gene family member A, Cdc42, integrin, integrin-linked kinase, and vascular endothelial growth factor signaling in DKD glomeruli. The tubulointerstitial compartment showed strong enrichment for inflammation-related pathways. The canonical complement signaling pathway was determined to be statistically differentially regulated in both DKD glomeruli and tubuli and was associated with increased glomerulosclerosis even in a different set of DKD samples. CONCLUSIONS Our studies have cataloged gene-expression regulation and identified multiple novel genes and pathways that may play a role in the pathogenesis of DKD or could serve as biomarkers. PMID:21752957

The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. Here we apply in vivo RNA crosslinking (CRAC) to the nucleases (Rrp44, Rrp6), two structural subunits (Rrp41, Csl4) and a cofactor (Trf4) of the yeast exosome. Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of non-coding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome indicating that substrates follow multiple pathways to the nucleases. PMID:23000172

Hermaphroditic flowers have evolved primarily under the selection on male function. Evolutionary modification often leads to stamen differentiation within flowers, or “heteranthery”, a phenomenon intrigued scientists since the 18th century until recently. However, the genetic basis and molecular regulation mechanism has barely been touched. Here we conducted comparative transcriptome profiling in Cassia biscapsularis L., a heterantherous species with representative patterns of stamen differentiation. Numerous differentially expressed genes (DEGs) were detected between the staminodes (the degenerated stamens) and fertile stamens, while much fewer genes differentially expressed among the three sets of fertile stamens. GO term enrichment and KEGG pathway analysis characterized functional properties of DEGs in different stamen types. Transcripts showing close correlation between expression pattern and stamen types were identified. Transcription factors from the bHLH family were suggested to have taken crucial part in the formation of staminodes. This first global transcriptomicanalysis focusing on stamen differentiation opens the door toward a more comprehensive understanding on the molecular regulation of floral organ evolution. Especially, the generated unigene resource would be valuable for developing male sterile lines in agronomy. PMID:27527392

Hermaphroditic flowers have evolved primarily under the selection on male function. Evolutionary modification often leads to stamen differentiation within flowers, or "heteranthery", a phenomenon intrigued scientists since the 18(th) century until recently. However, the genetic basis and molecular regulation mechanism has barely been touched. Here we conducted comparative transcriptome profiling in Cassia biscapsularis L., a heterantherous species with representative patterns of stamen differentiation. Numerous differentially expressed genes (DEGs) were detected between the staminodes (the degenerated stamens) and fertile stamens, while much fewer genes differentially expressed among the three sets of fertile stamens. GO term enrichment and KEGG pathway analysis characterized functional properties of DEGs in different stamen types. Transcripts showing close correlation between expression pattern and stamen types were identified. Transcription factors from the bHLH family were suggested to have taken crucial part in the formation of staminodes. This first global transcriptomicanalysis focusing on stamen differentiation opens the door toward a more comprehensive understanding on the molecular regulation of floral organ evolution. Especially, the generated unigene resource would be valuable for developing male sterile lines in agronomy. PMID:27527392

Plant's response and adaptation to abiotic stresses involve sophisticated genetic and epigenetic regulatory systems. To obtain a global view of molecular response to osmotic stresses, including the non-coding portion of genome, we conducted a total leaf transcriptomeanalysis on maize plants subjected to prolonged drought and salt stresses. Stress application to both B73 wild type and the epiregulator mutant rpd1-1/rmr6 allowed dissection of the epigenetic component of stress response. Coupling total RNA-Seq and transcriptome re-assembly we annotated thousands of new maize transcripts, together with 13,387 lncRNAs that may play critical roles in regulating gene expression. Differential expression analysis revealed hundreds of genes modulated by long-term stress application, including also many lncRNAs and transposons specifically induced by stresses. The amplitude and dynamic of the stress-modulated gene sets are very different between B73 and rpd1-1/rmr6 mutant plants, as result of stress-like effect on genome regulation caused by the mutation itself, which activates many stress-related genes even in control condition. The analyzed extensive set of total RNA-Seq data, together with the improvement of the transcriptome and the identification of the non-coding portion of the transcriptome give a revealing insight into the genetic and epigenetic mechanism responsible for maize molecular response to abiotic stresses. PMID:27461139

Plant’s response and adaptation to abiotic stresses involve sophisticated genetic and epigenetic regulatory systems. To obtain a global view of molecular response to osmotic stresses, including the non-coding portion of genome, we conducted a total leaf transcriptomeanalysis on maize plants subjected to prolonged drought and salt stresses. Stress application to both B73 wild type and the epiregulator mutant rpd1-1/rmr6 allowed dissection of the epigenetic component of stress response. Coupling total RNA-Seq and transcriptome re-assembly we annotated thousands of new maize transcripts, together with 13,387 lncRNAs that may play critical roles in regulating gene expression. Differential expression analysis revealed hundreds of genes modulated by long-term stress application, including also many lncRNAs and transposons specifically induced by stresses. The amplitude and dynamic of the stress-modulated gene sets are very different between B73 and rpd1-1/rmr6 mutant plants, as result of stress-like effect on genome regulation caused by the mutation itself, which activates many stress-related genes even in control condition. The analyzed extensive set of total RNA-Seq data, together with the improvement of the transcriptome and the identification of the non-coding portion of the transcriptome give a revealing insight into the genetic and epigenetic mechanism responsible for maize molecular response to abiotic stresses. PMID:27461139

Background The chemical components of sex pheromones have been determined for more than a thousand moth species, but so far only a handful of genes encoding enzymes responsible for the biosynthesis of these compounds have been identified. For understanding the evolution of moth sexual communication, it is essential to know which genes are involved in the production of specific pheromone components and what controls the variation in their relative frequencies in the pheromone blend. We used a transcriptomic approach to characterize the pheromone gland of the Noctuid moth Heliothis virescens, an important agricultural pest, in order to obtain substantial general sequence information and to identify a range of candidate genes involved in the pheromone biosynthetic pathway. Results To facilitate identifying sets of genes involved in a broad range of processes and to capture rare transcripts, we developed our majority of ESTs from a normalized cDNA library of Heliothis virescens pheromone glands (PG). Combining these with a non-normalized library yielded a total of 17,233 ESTs, which assembled into 2,082 contigs and 6,228 singletons. Using BLAST searches of the NR and Swissprot databases we were able to identify a large number of putative unique gene elements (unigenes), which we compared to those derived from previous transcriptomic surveys of the larval stage of Heliothis virescens. The distribution of unigenes among GO Biological Process functional groups shows an overall similarity between PG and larval transcriptomes, but with distinct enrichment of specific pathways in the PG. In addition, we identified a large number of candidate genes in the pheromone biosynthetic pathways. Conclusion These data constitute one of the first large-scale EST-projects for Noctuidae, a much-needed resource for exploring these pest species. Our analysis shows a surprisingly complex transcriptome and we identified a large number of potential pheromone biosynthetic pathway and immune

The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps. As major contributors to head regeneration, we identified diverse signaling pathways adopted for the regeneration response as well as enriched novel genes. Our global analysis reveals two distinct molecular cascades: an early injury response and a subsequent, signaling driven patterning of the regenerating tissue. A key factor of the initial injury response is a general stabilization of proteins and a net upregulation of transcripts, which is followed by a subsequent activation cascade of signaling molecules including Wnts and transforming growth factor (TGF) beta-related factors. We observed moderate overlap between the factors contributing to proteomic and transcriptomic responses suggesting a decoupled regulation between the transcriptional and translational levels. Our data also indicate that interstitial stem cells and their derivatives (e.g., neurons) have no major role in Hydra head regeneration. Remarkably, we found an enrichment of evolutionarily more recent genes in the early regeneration response, whereas conserved genes are more enriched in the late phase. In addition, genes specific to the early injury response were enriched in transposon insertions. Genetic dynamicity and taxon-specific factors might therefore play a hitherto underestimated role in Hydra regeneration. PMID:25841488

Paulownia fortunei is an ecologically and economically important tree species that is widely used as timber and chemical pulp. Its autotetraploid, which carries a number of valuable traits, was successfully induced with colchicine. To identify differences in gene expression between P. fortunei and its synthesized autotetraploid, we performed transcriptome sequencing using an Illumina Genome Analyzer IIx (GAIIx). About 94.8 million reads were generated and assembled into 383,056 transcripts, including 18,984 transcripts with a complete open reading frame. A conducted Basic Local Alignment Search Tool (BLAST) search indicated that 16,004 complete transcripts had significant hits in the National Center for Biotechnology Information (NCBI) non-redundant database. The complete transcripts were given functional assignments using three public protein databases. One thousand one hundred fifty eight differentially expressed complete transcripts were screened through a digital abundance analysis, including transcripts involved in energy metabolism and epigenetic regulation. Finally, the expression levels of several transcripts were confirmed by quantitative real-time PCR. Our results suggested that polyploidization caused epigenetic-related changes, which subsequently resulted in gene expression variation between diploid and autotetraploid P. fortunei. This might be the main mechanism affected by the polyploidization. Our results represent an extensive survey of the P. fortunei transcriptome and will facilitate subsequent functional genomics research in P. fortunei. Moreover, the gene expression profiles of P. fortunei and its autopolyploid will provide a valuable resource for the study of polyploidization. PMID:24663058

Paulownia fortunei is an ecologically and economically important tree species that is widely used as timber and chemical pulp. Its autotetraploid, which carries a number of valuable traits, was successfully induced with colchicine. To identify differences in gene expression between P. fortunei and its synthesized autotetraploid, we performed transcriptome sequencing using an Illumina Genome Analyzer IIx (GAIIx). About 94.8 million reads were generated and assembled into 383,056 transcripts, including 18,984 transcripts with a complete open reading frame. A conducted Basic Local Alignment Search Tool (BLAST) search indicated that 16,004 complete transcripts had significant hits in the National Center for Biotechnology Information (NCBI) non-redundant database. The complete transcripts were given functional assignments using three public protein databases. One thousand one hundred fifty eight differentially expressed complete transcripts were screened through a digital abundance analysis, including transcripts involved in energy metabolism and epigenetic regulation. Finally, the expression levels of several transcripts were confirmed by quantitative real-time PCR. Our results suggested that polyploidization caused epigenetic-related changes, which subsequently resulted in gene expression variation between diploid and autotetraploid P. fortunei. This might be the main mechanism affected by the polyploidization. Our results represent an extensive survey of the P. fortunei transcriptome and will facilitate subsequent functional genomics research in P. fortunei. Moreover, the gene expression profiles of P. fortunei and its autopolyploid will provide a valuable resource for the study of polyploidization. PMID:24663058

The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps. As major contributors to head regeneration, we identified diverse signaling pathways adopted for the regeneration response as well as enriched novel genes. Our global analysis reveals two distinct molecular cascades: an early injury response and a subsequent, signaling driven patterning of the regenerating tissue. A key factor of the initial injury response is a general stabilization of proteins and a net upregulation of transcripts, which is followed by a subsequent activation cascade of signaling molecules including Wnts and transforming growth factor (TGF) beta-related factors. We observed moderate overlap between the factors contributing to proteomic and transcriptomic responses suggesting a decoupled regulation between the transcriptional and translational levels. Our data also indicate that interstitial stem cells and their derivatives (e.g., neurons) have no major role in Hydra head regeneration. Remarkably, we found an enrichment of evolutionarily more recent genes in the early regeneration response, whereas conserved genes are more enriched in the late phase. In addition, genes specific to the early injury response were enriched in transposon insertions. Genetic dynamicity and taxon-specific factors might therefore play a hitherto underestimated role in Hydra regeneration. PMID:25841488

Because human epidermal melanocytes (HEMs) provide critical protection against skin cancer, sunburn, and photoaging, a genome-wide perspective of gene expression in these cells is vital to understanding human skin physiology. In this study we performed high throughput sequencing of HEMs to obtain a complete data set of transcript sizes, abundances, and splicing. As expected, we found that melanocyte specific genes that function in pigmentation were among the highest expressed genes. We analyzed receptor, ion channel and transcription factor gene families to get a better understanding of the cell signalling pathways used by melanocytes. We also performed a comparative transcriptomicanalysis of lightly versus darkly pigmented HEMs and found 16 genes differentially expressed in the two pigmentation phenotypes; of those, only one putative melanosomal transporter (SLC45A2) has known function in pigmentation. In addition, we found 166 genes with splice isoforms expressed exclusively in one pigmentation phenotype, 17 of which are genes involved in signal transduction. Our melanocyte transcriptome study provides a comprehensive view and may help identify novel pigmentation genes and potential pharmacological targets. PMID:25451175

STUDY QUESTION How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptomeanalysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603

RNA is a polymeric molecule implicated in various biological processes, such as the coding, decoding, regulation, and expression of genes. Numerous studies have examined RNA features using whole transcriptome sequencing (RNA-seq) approaches. RNA-seq is a powerful technique for characterizing and quantifying the transcriptome and accelerates the development of bioinformatics software. In this review, we introduce routine RNA-seq workflow together with related software, focusing particularly on transcriptome reconstruction and expression quantification. PMID:26865842

RNA is a polymeric molecule implicated in various biological processes, such as the coding, decoding, regulation, and expression of genes. Numerous studies have examined RNA features using whole transcriptome sequencing (RNA-seq) approaches. RNA-seq is a powerful technique for characterizing and quantifying the transcriptome and accelerates the development of bioinformatics software. In this review, we introduce routine RNA-seq workflow together with related software, focusing particularly on transcriptome reconstruction and expression quantification. PMID:26865842

Custom sequence capture experiments are becoming an efficient approach for gathering large sets of orthologous markers in nonmodel organisms. Transcriptome-based exon capture utilizes transcript sequences to design capture probes, typically using a reference genome to identify intron-exon boundaries to exclude shorter exons (<200 bp). Here, we test directly using transcript sequences for probe design, which are often composed of multiple exons of varying lengths. Using 1260 orthologous transcripts, we conducted sequence captures across multiple phylogenetic scales for frogs, including outgroups ~100 Myr divergent from the ingroup. We recovered a large phylogenomic data set consisting of sequence alignments for 1047 of the 1260 transcriptome-based loci (~561 000 bp) and a large quantity of highly variable regions flanking the exons in transcripts (~70 000 bp), the latter improving substantially by only including ingroup species (~797 000 bp). We recovered both shorter (<100 bp) and longer exons (>200 bp), with no major reduction in coverage towards the ends of exons. We observed significant differences in the performance of blocking oligos for target enrichment and nontarget depletion during captures, and differences in PCR duplication rates resulting from the number of individuals pooled for capture reactions. We explicitly tested the effects of phylogenetic distance on capture sensitivity, specificity, and missing data, and provide a baseline estimate of expectations for these metrics based on a priori knowledge of nuclear pairwise differences among samples. We provide recommendations for transcriptome-based exon capture design based on our results, cost estimates and offer multiple pipelines for data assembly and analysis. PMID:27241806

Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. To characterize the citrate utilization in C. glutamicum on a genomewide scale, a comparative analysis was carried out by combining transcriptome and proteome analysis. In cells grown on citrate, transcriptomeanalysis revealed highest expression changes for two different citrate-uptake systems encoded by citM and tctCBA, whereas genes encoding uptake systems for the glucose- (ptsG), sucrose- (ptsS) and fructose- (ptsF) specific PTS components and permeases for gluconate (gntP) and glutamate (gluC) displayed decreased mRNA levels in citrate-grown cells. This pattern was also observed when cells grown in Luria-Bertani (LB) medium plus citrate were compared with cells grown in LB medium, indicating some kind of catabolite repression. Genes encoding enzymes of the tricarboxylic acid cycle (aconitase, succinyl-CoA synthetase, succinate dehydrogenase and fumarase), malic enzyme, PEP carboxykinase, gluconeogenic glyceraldehyde-3-phosphate dehydrogenase and ATP synthase displayed increased expression in cells grown on citrate. Accordingly, proteome analysis revealed elevated protein levels of these enzymes and showed a good correlation with the mRNA levels. In conclusion, this study revealed the citrate stimulon in C. glutamicum and the regulated central metabolic genes when grown on citrate. PMID:17559405

One of the central issues in evolutionary developmental biology is how we can formulate the relationships between evolutionary and developmental processes. Two major models have been proposed: the 'funnel-like' model, in which the earliest embryo shows the most conserved morphological pattern, followed by diversifying later stages, and the 'hourglass' model, in which constraints are imposed to conserve organogenesis stages, which is called the phylotypic period. Here we perform a quantitative comparative transcriptomeanalysis of several model vertebrate embryos and show that the pharyngula stage is most conserved, whereas earlier and later stages are rather divergent. These results allow us to predict approximate developmental timetables between different species, and indicate that pharyngula embryos have the most conserved gene expression profiles, which may be the source of the basic body plan of vertebrates. PMID:21427719

Ovary development is a complex process involving numerous genes. A well-developed ovary is essential for females to keep fertility and reproduce offspring. In order to gain a better insight into the molecular mechanisms related to the process of mammalian ovary development, we performed a comparative transcriptomicanalysis on ovaries isolated from infant and adult mice by using next-generation sequencing technology (SOLiD). We identified 15,454 and 16,646 transcriptionally active genes at the infant and adult stage, respectively. Among these genes, we also identified 7021 differentially expressed genes. Our analysis suggests that, in general, the adult ovary has a higher level of transcriptomic activity. However, it appears that genes related to primordial follicle development, such as those encoding Figla and Nobox, are more active in the infant ovary, whereas expression of genes vital for follicle development, such as Gdf9, Bmp4 and Bmp15, is upregulated in the adult. These data suggest a dynamic shift in gene expression during ovary development and it is apparent that these changes function to facilitate follicle maturation, when additional functional gene studies are considered. Furthermore, our investigation has also revealed several important functional pathways, such as apoptosis, MAPK and steroid biosynthesis, that appear to be much more active in the adult ovary compared to those of the infant. These findings will provide a solid foundation for future studies on ovary development in mice and other mammals and help to expand our understanding of the complex molecular and cellular events that occur during postnatal ovary development. PMID:25251848

Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., a relic tree of the Tertiary period, plays a critical role in maintaining desert ecosystems in the Mid-Asia region. Genome-scale gene expression profiling studies will provide deep insight into the molecular mechanism underlying the drought tolerance of A. mongolicus. In the present study, we investigated the transcriptional changes induced by drought treatment in A. mongolicus leaves by establishing a comprehensive transcriptome database and then performing a Digital Gene Expression (DGE) analysis using Solexa sequencing technology. A comprehensive transcriptome database was obtained by assembling the Illumina unigenes with expressed sequence tags (EST) available publicly, and other high throughput sequencing data. To analyze the dynamic and complicated gene regulation network during PEG6000-induced drought treatment in leaves of A. mongolicus, a time-course gene expression analysis was performed using tag-based DGE technology, which identified 437, 1,247 and 802 differentially expressed transcripts in 1, 24 and 72 h drought stress libraries, respectively. GO and KEGG analyses revealed hormone signal transduction and phenylpropanoid biosynthesis were enriched during drought treatment. A batch of drought-regulated transcription factor transcripts were identified, including the subsets of HD-ZIP, bZIP, WRKY, AP2/ERF and bHLH family members, which may play roles in drought response in A. mongolicus. The sequence collection assembled in the present study represents one of the most comprehensive transcriptome databases for A. mongolicus currently. The differentially expressed transcripts identified in our study provide a good start for identifying the key genes in stress response and performing functional analysis to reveal their roles in stress adaptation in planta. PMID:25923822

Ammopiptanthus mongolicus (Maxim. Ex Kom.) Cheng f., a relic tree of the Tertiary period, plays a critical role in maintaining desert ecosystems in the Mid-Asia region. Genome-scale gene expression profiling studies will provide deep insight into the molecular mechanism underlying the drought tolerance of A. mongolicus. In the present study, we investigated the transcriptional changes induced by drought treatment in A. mongolicus leaves by establishing a comprehensive transcriptome database and then performing a Digital Gene Expression (DGE) analysis using Solexa sequencing technology. A comprehensive transcriptome database was obtained by assembling the Illumina unigenes with expressed sequence tags (EST) available publicly, and other high throughput sequencing data. To analyze the dynamic and complicated gene regulation network during PEG6000-induced drought treatment in leaves of A. mongolicus, a time-course gene expression analysis was performed using tag-based DGE technology, which identified 437, 1,247 and 802 differentially expressed transcripts in 1, 24 and 72 h drought stress libraries, respectively. GO and KEGG analyses revealed hormone signal transduction and phenylpropanoid biosynthesis were enriched during drought treatment. A batch of drought-regulated transcription factor transcripts were identified, including the subsets of HD-ZIP, bZIP, WRKY, AP2/ERF and bHLH family members, which may play roles in drought response in A. mongolicus. The sequence collection assembled in the present study represents one of the most comprehensive transcriptome databases for A. mongolicus currently. The differentially expressed transcripts identified in our study provide a good start for identifying the key genes in stress response and performing functional analysis to reveal their roles in stress adaptation in planta. PMID:25923822

Snakebite in Africa causes thousands of deaths annually and considerable permanent physical disability. The saw-scaled viper, Echis ocellatus, represents the single most medically important snake species in West Africa. To provide a detailed compositional analysis of the venom of E. ocellatus for designing novel toxin-specific immunotherapy and to delineate sequence structure-function relationships of individual toxins, we characterised the venom proteome and the venom gland transcriptome. Whole E. ocellatus venom was fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction using a combination of SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and CID-MS/MS of tryptic peptides. This analysis identified around 35 distinct proteins of molecular masses in the range of 5.5-110 kDa belonging to 8 different toxin families (disintegrin, DC-fragment, phospholipase A(2), cysteine-rich secretory protein, serine proteinase, C-type lectin, l-amino acid oxidase, and Zn(2+)-dependent metalloprotease). Comparison of the toxin composition of E. ocellatus venom determined using a proteomic approach, with the predicted proteome derived from assembly of 1000 EST sequences from a E. ocellatus venom gland cDNA library, shows some differences. Most notably, peptides derived from 26% of the venom proteins could not be ascribed an exact match in the transcriptome. Similarly, 64 (67%) out of the 95 putative toxin clusters reported in the transcriptome did not match to peptides detected in the venom proteome. These data suggest that the final composition of venom is influenced by transcriptional and post-translational mechanisms that may be more complex than previously appreciated. This, in turn, emphasises the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the precise composition of snake venom, than would be gleaned from using one analysis alone. From a clinical perspective, the large

Background In trying to understand the evolutionary relationships of organisms, the current flood of sequence data offers great opportunities, but also reveals new challenges with regard to data quality, the selection of data for subsequent analysis, and the automation of steps that were once done manually for single-gene analyses. Even though genome or transcriptome data is available for representatives of most bilaterian phyla, some enigmatic taxa still have an uncertain position in the animal tree of life. This is especially true for myzostomids, a group of symbiotic (or parasitic) protostomes that are either placed with annelids or flatworms. Methodology Based on similarity criteria, Illumina-based transcriptome sequences of one myzostomid were compared to protein sequences of one additional myzostomid and 29 reference metazoa and clustered into gene families. These families were then used to investigate the phylogenetic position of Myzostomida using different approaches: Alignments of 989 sequence families were concatenated, and the resulting superalignment was analyzed under a Maximum Likelihood criterion. We also used all 1,878 gene trees with at least one myzostomid sequence for a supertree approach: the individual gene trees were computed and then reconciled into a species tree using gene tree parsimony. Conclusions Superalignments require strictly orthologous genes, and both the gene selection and the widely varying amount of data available for different taxa in our dataset may cause anomalous placements and low bootstrap support. In contrast, gene tree parsimony is designed to accommodate multilocus gene families and therefore allows a much more comprehensive data set to be analyzed. Results of this supertree approach showed a well-resolved phylogeny, in which myzostomids were part of the annelid radiation, and major bilaterian taxa were found to be monophyletic. PMID:22276131

Pangolins are scale-covered mammals, containing eight endangered species. Maintaining pangolins in captivity is a significant challenge, in part because little is known about their genetics. Here we provide the first large-scale sequencing of the critically endangered Manis javanica transcriptomes from eight different organs using Illumina HiSeq technology, yielding ~75 Giga bases and 89,754 unigenes. We found some unigenes involved in the insect hormone biosynthesis pathway and also 747 lipids metabolism-related unigenes that may be insightful to understand the lipid metabolism system in pangolins. Comparative analysis between M. javanica and other mammals revealed many pangolin-specific genes significantly over-represented in stress-related processes, cell proliferation and external stimulus, probably reflecting the traits and adaptations of the analyzed pregnant female M. javanica. Our study provides an invaluable resource for future functional works that may be highly relevant for the conservation of pangolins. PMID:27618997

Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus), a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB) transcriptome, a number of aminopeptidase N (APN) cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptomeanalysis will certainly be useful to identify novel developmental genes, to better understand the insect’s biology and to guide the development of new strategies for insect-pest control. PMID:25706301

Angiosperms are renown for their diversity of flower colors. Often considered adaptations to pollinators, the most common underlying pigments, anthocyanins, are also involved in plants' stress response. Although the anthocyanin biosynthetic pathway is well characterized across many angiosperms and is composed of a few candidate genes, the consequences of blocking this pathway and producing white flowers has not been investigated at the transcriptomescale. We take a transcriptome-wide approach to compare expression differences between purple and white petal buds in the arctic mustard, Parrya nudicaulis, to determine which genes' expression are consistently correlated with flower color. Using mRNA-Seq and de novo transcriptome assembly, we assembled an average of 722 bp per gene (49.81% coding sequence based on the A. thaliana homolog) for 12,795 genes from the petal buds of a pair of purple and white samples. Our results correlate strongly with qRT-PCR analysis of nine candidate genes in the anthocyanin biosynthetic pathway where chalcone synthase has the greatest difference in expression between color morphs (P/W = ∼7×). Among the most consistently differentially expressed genes between purple and white samples, we found 3× more genes with higher expression in white petals than in purple petals. These include four unknown genes, two drought-response genes (CDSP32, ERD5), a cold-response gene (GR-RBP2), and a pathogen defense gene (DND1). Gene ontology analysis of the top 2% of genes with greater expression in white relative to purple petals revealed enrichment in genes associated with stress responses including cold, drought and pathogen defense. Unlike the uniform downregulation of chalcone synthase that may be directly involved in the loss of petal anthocyanins, the variable expression of several genes with greater expression in white petals suggest that the physiological and ecological consequences of having white petals may be microenvironment

Transcriptional profiling is a powerful approach to study mouse development, physiology, and disease models. Here, we describe a protocol for mouse thiouracil-tagging (TU-tagging), a transcriptomeanalysis technology that includes in vivo covalent labeling, purification, and analysis of cell type-specific RNA. TU-tagging enables 1) the isolation of RNA from a given cell population of a complex tissue, avoiding transcriptional changes induced by cell isolation trauma, and 2) the identification of actively transcribed RNAs and not pre-existing transcripts. Therefore, in contrast to other cell-specific transcriptional profiling methods based on purification of tagged ribosomes or nuclei, TU-tagging provides a direct examination of transcriptional regulation. We describe how to: 1) deliver 4-thiouracil to transgenic mice to thio-label cell lineage-specific transcripts, 2) purify TU-tagged RNA and prepare libraries for Illumina sequencing, and 3) follow a straight-forward bioinformatics workflow to identify cell type-enriched or differentially expressed genes. Tissue containing TU-tagged RNA can be obtained in one day, RNA-Seq libraries generated within two days, and, following sequencing, an initial bioinformatics analysis completed in one additional day. PMID:24457332

Embryonic stem (ES) cells have a remarkable capacity to self-organize complex, multi-layered optic cups in vitro via a culture technique called SFEBq. During both SFEBq and in vivo optic cup development, Rax (Rx) expressing neural retina epithelial (NRE) tissues utilize Fgf and Wnt/β-catenin signalling pathways to differentiate into neural retina (NR) and retinal-pigmented epithelial (RPE) tissues, respectively. How these signaling pathways affect gene expression during optic tissue formation has remained largely unknown, especially at the transcriptomescale. Here, we address this question using RNA-Seq. We generated Rx+ optic tissue using SFEBq, exposed these tissues to either Fgf or Wnt/β-catenin stimulation, and assayed their gene expression across multiple time points using RNA-Seq. This comparative dataset will help elucidate how Fgf and Wnt/β-catenin signaling affect gene expression during optic tissue differentiation and will help inform future efforts to optimize in vitro optic tissue culture technology. PMID:26110066

Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family. PMID:26743902

Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family. PMID:26743902

IMPORTANCE The core clinical and neuropathological feature of the autosomal dominant spinocerebellar ataxias (SCAs) is cerebellar degeneration. Mutations in the known genes explain only 50% to 60% of SCA cases. To date, no effective treatments exist, and the knowledge of drug-treatable molecular pathways is limited. The examination of overlapping mechanisms and the interpretation of how ataxia genes interact will be important in the discovery of potential disease-modifying agents. OBJECTIVES To address the possible relationships among known SCA genes, predict their functions, identify overlapping pathways, and provide a framework for candidate gene discovery using whole-transcriptome expression data. DESIGN, SETTING, AND PARTICIPANTS We have used a systems biology approach based on whole-transcriptome gene expression analysis. As part of the United Kingdom Brain Expression Consortium, we analyzed the expression profile of 788 brain samples obtained from 101 neuropathologically healthy individuals (10 distinct brain regions each). Weighted gene coexpression network analysis was used to cluster 24 SCA genes into gene coexpression modules in an unsupervised manner. The overrepresentation of SCA transcripts in modules identified in the cerebellum was assessed. Enrichment analysis was performed to infer the functions and molecular pathways of genes in biologically relevant modules. MAIN OUTCOMES AND MEASURES Molecular functions and mechanisms implicating SCA genes, as well as lists of relevant coexpressed genes as potential candidates for novel SCA causative or modifier genes. RESULTS Two cerebellar gene coexpression modules were statistically enriched in SCA transcripts (P = .021 for the tan module and P = 2.87 × 10−5 for the light yellow module) and contained established granule and Purkinje cell markers, respectively. One module includes genes involved in the ubiquitin-proteasome system and contains SCA genes usually associated with a complex phenotype, while the

Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptomeanalysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses. PMID:26710886

Background Common bean (Phaseolus vulgaris) is the most important food legume in the world. Although this crop is very important to both the developed and developing world as a means of dietary protein supply, resources available in common bean are limited. Global transcriptomeanalysis is important to better understand gene expression, genetic variation, and gene structure annotation in addition to other important features. However, the number and description of common bean sequences are very limited, which greatly inhibits genome and transcriptome research. Here we used 454 pyrosequencing to obtain a substantial transcriptome dataset for common bean. Results We obtained 1,692,972 reads with an average read length of 207 nucleotides (nt). These reads were assembled into 59,295 unigenes including 39,572 contigs and 19,723 singletons, in addition to 35,328 singletons less than 100 bp. Comparing the unigenes to common bean ESTs deposited in GenBank, we found that 53.40% or 31,664 of these unigenes had no matches to this dataset and can be considered as new common bean transcripts. Functional annotation of the unigenes carried out by Gene Ontology assignments from hits to Arabidopsis and soybean indicated coverage of a broad range of GO categories. The common bean unigenes were also compared to the bean bacterial artificial chromosome (BAC) end sequences, and a total of 21% of the unigenes (12,724) including 9,199 contigs and 3,256 singletons match to the 8,823 BAC-end sequences. In addition, a large number of simple sequence repeats (SSRs) and transcription factors were also identified in this study. Conclusions This work provides the first large scale identification of the common bean transcriptome derived by 454 pyrosequencing. This research has resulted in a 150% increase in the number of Phaseolus vulgaris ESTs. The dataset obtained through this analysis will provide a platform for functional genomics in common bean and related legumes and will aid in the

In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12-30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq) technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25) by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs) revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation). This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy. PMID:26703736

With the increasing amount of data produced by high-throughput technologies in many fields of science, clustering has become an integral step in exploratory data analysis in order to group similar elements into classes. However, many clustering algorithms can only work properly if aided by human expertise. For example, one parameter which is crucial and often manually set is the number of clusters present in the analyzed set. We present a novel stopping rule to find the optimal number of clusters based on the comparison of the density of points inside the clusters and between them. The method is evaluated on synthetic as well as on real transcriptomic data and compared with two current methods. Finally, we illustrate its usefulness in the analysis of the expression profiles of promyelocytic cells before and after treatment with all-trans retinoic acid. Simultaneous clustering for gene regulation and absolute initial expression levels allowed the identification of numerous genes associated with signal transduction revealing the complexity of retinoic acid signaling. PMID:12235383

The wild silkworm Bombyx mandarina is widely believed to be an ancestor of the domesticated silkworm, Bombyx mori. Silkworms are often used as a model for studying the mechanism of species domestication. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform. We produced 100,004,078 high-quality reads and assembled them into 50,773 contigs with an N50 length of 1764 bp and a mean length of 941.62 bp. A total of 33,759 unigenes were identified, with 12,805 annotated in the Nr database, 8273 in the Pfam database, and 9093 in the Swiss-Prot database. Expression profile analysis found significant differential expression of 1308 unigenes between the middle silk gland (MSG) and posterior silk gland (PSG). Three sericin genes (sericin 1, sericin 2, and sericin 3) were expressed specifically in the MSG and three fibroin genes (fibroin-H, fibroin-L, and fibroin/P25) were expressed specifically in the PSG. In addition, 32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. Comparison with the domesticated silkworm p50/Dazao identified 5,295 orthologous genes, among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research. PMID:25806526

Nasopharyngeal carcinoma (NPC) is a malignant cancer arising from the epithelial surface of the nasopharynx that mostly appears in advanced stages of the disease, leading to a poor prognosis. To date, a number of mRNA profiling investigations on NPC have been reported in order to identify suitable biomarkers for early detection. However, the results may be specific to each study with distinct sample types. In this study, an integrative meta-analysis of NPC transcriptome data was performed to determine dysregulated pathways, potentially leading to identification of molecular markers. Ten independent NPC gene expression profiling microarray datasets, including 135 samples from NPC cell lines, primary cell lines, and tissues were assimilated into a meta-analysis and cross-validation to identify a cohort of genes that were significantly dysregulated in NPC. Bioinformatics analyses of these genes revealed the significant pathways and individual players involving in cellular metabolism, cell cycle regulation, DNA repair, as well as ErbB pathway. Altogether, we propose that dysregulation of these molecular pathways in NPC might play a role in the NPC pathogenesis, providing clues, which could eventually translate into diagnostic and therapeutic approaches. PMID:25724187

Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as MPV-17, REV1 and HSP68. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis. PMID:26274803

In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12–30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq) technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25) by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs) revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation). This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy. PMID:26703736

The wild silkworm Bombyx mandarina is widely believed to be an ancestor of the domesticated silkworm, Bombyx mori. Silkworms are often used as a model for studying the mechanism of species domestication. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform. We produced 100,004,078 high-quality reads and assembled them into 50,773 contigs with an N50 length of 1764 bp and a mean length of 941.62 bp. A total of 33,759 unigenes were identified, with 12,805 annotated in the Nr database, 8273 in the Pfam database, and 9093 in the Swiss-Prot database. Expression profile analysis found significant differential expression of 1308 unigenes between the middle silk gland (MSG) and posterior silk gland (PSG). Three sericin genes (sericin 1, sericin 2, and sericin 3) were expressed specifically in the MSG and three fibroin genes (fibroin-H, fibroin-L, and fibroin/P25) were expressed specifically in the PSG. In addition, 32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. Comparison with the domesticated silkworm p50/Dazao identified 5,295 orthologous genes, among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research. PMID:25806526

Understanding the molecular interactions that lead to the establishment of the major body axes during embryogenesis is one of the main goals of developmental biology. Although the past two decades have revolutionized our knowledge about the genetic basis of these patterning processes, the list of genes involved in axis formation is unlikely to be complete. In order to identify new genes involved in the establishment of the dorsoventral (DV) axis during early stages of zebrafish embryonic development, we employed next generation sequencing for full transcriptomeanalysis of normal embryos and embryos lacking overt DV pattern. A combination of different statistical approaches yielded 41 differentially expressed candidate genes and we confirmed by in situ hybridization the early dorsal expression of 32 genes that are transcribed shortly after the onset of zygotic transcription. Although promoter analysis of the validated genes suggests no general enrichment for the binding sites of early acting transcription factors, most of these genes carry “bivalent” epigenetic histone modifications at the time when zygotic transcription is initiated, suggesting a “poised” transcriptional status. Our results reveal some new candidates of the dorsal gene regulatory network and suggest that a plurality of the earliest upregulated genes on the dorsal side have a role in the modulation of the canonical Wnt pathway. PMID:23922899

Background Comprehensive, tissue-specific, microarray analysis is a potent tool for the identification of tightly defined expression patterns that might be missed in whole-organism scans. We applied such an analysis to Drosophila melanogaster Malpighian (renal) tubule, a defined differentiated tissue. Results The transcriptome of the D. melanogaster Malpighian tubule is highly reproducible and significantly different from that obtained from whole-organism arrays. More than 200 genes are more than 10-fold enriched and over 1,000 are significantly enriched. Of the top 200 genes, only 18 have previously been named, and only 45% have even estimates of function. In addition, 30 transcription factors, not previously implicated in tubule development, are shown to be enriched in adult tubule, and their expression patterns respect precisely the domains and cell types previously identified by enhancer trapping. Of Drosophila genes with close human disease homologs, 50 are enriched threefold or more, and eight enriched 10-fold or more, in tubule. Intriguingly, several of these diseases have human renal phenotypes, implying close conservation of renal function across 400 million years of divergent evolution. Conclusions From those genes that are identifiable, a radically new view of the function of the tubule, emphasizing solute transport rather than fluid secretion, can be obtained. The results illustrate the phenotype gap: historically, the effort expended on a model organism has tended to concentrate on a relatively small set of processes, rather than on the spread of genes in the genome. PMID:15345053

Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as MPV-17, REV1 and HSP68. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis. PMID:26274803

To explore the transcriptome of Musca domestica larvae and to identify unique sequences, we used massively parallel pyrosequencing on the Roche 454-FLX platform to generate a substantial EST dataset of this fly. As a result, we obtained a total of 249,555 ESTs with an average read length of 373 bp. These reads were assembled into 13,206 contigs and 20,556 singletons. Using BlastX searches of the Swissprot and Nr databases, we were able to identify 4,814 contigs and 8,166 singletons as unique sequences. Subsequently, the annotated sequences were subjected to GO analysis and the search results showed a majority of the query sequences were assignable to certain gene ontology terms. In addition, functional classification and pathway assignment were performed by KEGG and 2,164 unique sequences were mapped into 184 KEGG pathways in total. As the first attempt on large-scale RNA sequencing of M. domestica, this general picture of the transcriptome can establish a fundamental resource for further research on functional genomics. PMID:21643958

Background A century ago, Chestnut Blight Disease (CBD) devastated the American chestnut. Backcross breeding has been underway to introgress resistance from Chinese chestnut into surviving American chestnut genotypes. Development of genomic resources for the family Fagaceae, has focused in this project on Castanea mollissima Blume (Chinese chestnut) and Castanea dentata (Marsh.) Borkh (American chestnut) to aid in the backcross breeding effort and in the eventual identification of blight resistance genes through genomic sequencing and map based cloning. A previous study reported partial characterization of the transcriptomes from these two species. Here, further analyses of a larger dataset and assemblies including both 454 and capillary sequences were performed and defense related genes with differential transcript abundance (GDTA) in canker versus healthy stem tissues were identified. Results Over one and a half million cDNA reads were assembled into 34,800 transcript contigs from American chestnut and 48,335 transcript contigs from Chinese chestnut. Chestnut cDNA showed higher coding sequence similarity to genes in other woody plants than in herbaceous species. The number of genes tagged, the length of coding sequences, and the numbers of tagged members within gene families showed that the cDNA dataset provides a good resource for studying the American and Chinese chestnut transcriptomes. In silico analysis of transcript abundance identified hundreds of GDTA in canker versus healthy stem tissues. A significant number of additional DTA genes involved in the defense-response not reported in a previous study were identified here. These DTA genes belong to various pathways involving cell wall biosynthesis, reactive oxygen species (ROS), salicylic acid (SA), ethylene, jasmonic acid (JA), abscissic acid (ABA), and hormone signalling. DTA genes were also identified in the hypersensitive response and programmed cell death (PCD) pathways. These DTA genes are candidates

Grapevine is one of the most economically important and widely cultivated fruit crop worldwide. With the industrialization and the popular application of cupric fungicides in grape industry, copper stress and copper pollution are also the factors affecting grape production and berry and wine quality. Here, 3,843 transcripts were significantly differently expressed genes in response to Cu stress by RNA-seq, which included 1,892 up-regulated and 1,951 down-regulated transcripts. During this study we found many known and novel Cu-induced and -repressed genes. Biological analysis of grape samples were indicated that exogenous Cu can influence chlorophylls metabolism and photosynthetic activities of grapevine. Most ROS detoxification systems, including antioxidant enzyme, stress-related proteins and secondary metabolites were strongly induced. Concomitantly, abscisic acid functioned as a negative regulator in Cu stress, in opposite action to ethylene, auxin, jasmonic acid, and brassinolide. This study also identified a set of Cu stress specifically activated genes coding copper transporter, P1B-type ATPase, multidrug transporters. Overall, this work was carried out to gain insights into the copper-regulated and stress-responsive mechanisms in grapevine at transcriptome level. This research can also provide some genetic information that can help us in better vinery management and breeding Cu-resistant grape cultivars. PMID:26673527

Grapevine is one of the most economically important and widely cultivated fruit crop worldwide. With the industrialization and the popular application of cupric fungicides in grape industry, copper stress and copper pollution are also the factors affecting grape production and berry and wine quality. Here, 3,843 transcripts were significantly differently expressed genes in response to Cu stress by RNA-seq, which included 1,892 up-regulated and 1,951 down-regulated transcripts. During this study we found many known and novel Cu-induced and -repressed genes. Biological analysis of grape samples were indicated that exogenous Cu can influence chlorophylls metabolism and photosynthetic activities of grapevine. Most ROS detoxification systems, including antioxidant enzyme, stress-related proteins and secondary metabolites were strongly induced. Concomitantly, abscisic acid functioned as a negative regulator in Cu stress, in opposite action to ethylene, auxin, jasmonic acid, and brassinolide. This study also identified a set of Cu stress specifically activated genes coding copper transporter, P1B-type ATPase, multidrug transporters. Overall, this work was carried out to gain insights into the copper-regulated and stress-responsive mechanisms in grapevine at transcriptome level. This research can also provide some genetic information that can help us in better vinery management and breeding Cu-resistant grape cultivars. PMID:26673527

To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum (Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and TICAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions. PMID:25091627

Plants are frequently exposed to microorganisms like fungi, bacteria, and viruses that cause biotic stresses. Fusarium head blight (FHB) is an economically risky wheat disease, which occurs upon Fusarium graminearum (Fg) infection. Moderately susceptible (cv. "Mizrak 98") and susceptible (cv. "Gun 91") winter type bread wheat cultivars were subjected to transcriptional profiling after exposure to Fg infection. To examine the early response to the pathogen in wheat, we measured gene expression alterations in mock and pathogen inoculated root crown of moderately susceptible (MS) and susceptible cultivars at 12 hours after inoculation (hai) using 12X135K microarray chip. The transcriptome analyses revealed that out of 39,179 transcripts, 3668 genes in microarray were significantly regulated at least in one time comparison. The majority of differentially regulated transcripts were associated with disease response and the gene expression mechanism. When the cultivars were compared, a number of transcripts and expression alterations varied within the cultivars. Especially membrane related transcripts were detected as differentially expressed. Moreover, diverse transcription factors showed significant fold change values among the cultivars. This study presented new insights to understand the early response of selected cultivars to the Fg at 12 hai. Through the KEGG analysis, we observed that the most altered transcripts were associated with starch and sucrose metabolism and gluconeogenesis pathways. PMID:26539199

Formation of the contractile myofibril of the skeletal muscle is a complex process which when perturbed leads to muscular dystrophy. Herein, we provide a mRNAseq dataset on three different zebrafish mutants affecting muscle organization during embryogenesis. These comprise the myosin folding chaperone unc45b (unc45b-/-), heat shock protein 90aa1.1 (hsp90aa1.1-/-) and the acetylcholine esterase (ache-/-) gene. The transcriptomeanalysis was performed in duplicate experiments at 72 h post-fertilization (hpf) for all three mutants, with two additional times of development (24 hpf and 48 hpf) for unc45b-/-. A total of 20 samples were analyzed by hierarchical clustering for differential gene expression. The data from this study support the observation made in Etard et al. (2015) [1] (http://dx.doi.org/10.1186/s13059-015-0825-8) that a failure to fold myosin activates a unique transcriptional program in the skeletal muscles that is different from that induced in stressed muscle cells. PMID:27274534

Population matrix models allow sets of demographic parameters to be summarized by a single value 8, the finite rate of population increase. The consequences of change in individual demographic parameters are naturally measured by the corresponding changes in 8; sensitivity analyses compare demographic parameters on the basis of these changes. These comparisons are complicated by issues of scale. Elasticity analysis attempts to deal with issues of scale by comparing the effects of proportional changes in demographic parameters, but leads to inconsistencies in evaluating demographic rates. We discuss this and other problems of scaling in sensitivity analysis, and suggest a simple criterion for choosing appropriate scales. We apply our suggestions to data for the killer whale, Orcinus orca.

Sterility is a serious problem that can affect all bionts. In teleosts, double haploids (DHs) induced by mitogynogenesis are often sterile. This sterility severely restricts the further application of DHs for production of clones, genetic analysis, and breeding. However, sterile DH individuals are good source materials for investigation of the molecular mechanisms of gonad development, especially for studies into the role of genes that are indispensable for fish reproduction. Here, we used the Illumina sequencing platform to analyze the transcriptome of sterile female DH Japanese flounder in order to identify major genes that cause sterility and to provide a molecular basis for an intensive study of gonadal development in teleosts. Through sequencing, assembly, and annotation, we obtained 52,474 contigs and found that 60.7% of these shared homologies with existing sequences. A total of 1225 differentially expressed unigenes were found, including 492 upregulated and 733 downregulated genes. Gene Ontology and KEGG analyses showed that genes showing significant upregulation, such as CYP11A1, CYP11B2, CYP17, CYP21, HSD3β, bcl2l1, and PRLR, principally correlated with sterol metabolic process, steroid biosynthetic process, and the Jak-stat signaling pathway. The significantly downregulated genes were primarily associated with immune response, antigen processing and presentation, cytokine–cytokine receptor interaction, and protein digestion and absorption. Using a co-expression network analysis, we conducted a comprehensive comparison of gene expression in the gonads of fertile and sterile female DH Japanese flounder. Identification of genes showing significantly different expression will provide further insights into DH reproductive dysfunction and oocyte maturation processes in teleosts. PMID:26580217

Soybean is an important crop for Brazilian agribusiness. However, many factors can limit its production, especially root-knot nematode infection. Studies on the mechanisms employed by the resistant soybean genotypes to prevent infection by these nematodes are of great interest for breeders. For these reasons, the aim of this work is to characterize the transcriptome of soybean line PI 595099-Meloidogyne javanica interaction through expression analysis. Two cDNA libraries were obtained using a pool of RNA from PI 595099 uninfected and M. javanica (J2) infected roots, collected at 6, 12, 24, 48, 96, 144 and 192 h after inoculation. Around 800 ESTs (Expressed Sequence Tags) were sequenced and clustered into 195 clusters. In silico subtraction analysis identified eleven differentially expressed genes encoding putative proteins sharing amino acid sequence similarities by using BlastX: metallothionein, SLAH4 (SLAC1 Homologue 4), SLAH1 (SLAC1 Homologue 1), zinc-finger proteins, AN1-type proteins, auxin-repressed proteins, thioredoxin and nuclear transport factor 2 (NTF-2). Other genes were also found exclusively in nematode stressed soybean roots, such as NAC domain-containing proteins, MADS-box proteins, SOC1 (suppressor of overexpression of constans 1) proteins, thioredoxin-like protein 4-Coumarate-CoA ligase and the transcription factor (TF) MYBZ2. Among the genes identified in non-stressed roots only were Ser/Thr protein kinases, wound-induced basic protein, ethylene-responsive family protein, metallothionein-like protein cysteine proteinase inhibitor (cystatin) and Putative Kunitz trypsin protease inhibitor. An understanding of the roles of these differentially expressed genes will provide insights into the resistance mechanisms and candidate genes involved in soybean-M. javanica interaction and contribute to more effective control of this pathogen. PMID:22802712

Termites are eusocial insects related to cockroaches that feed on lignocellulose. These insects are key species in ecosystems since they recycle a large amount of nutrients but also are pests, exerting major economic impacts. Knowledge on the molecular pathways underlying reproduction, caste differentiation or lignocellulose digestion would largely benefit from additional transcriptomic data. This study focused on transcriptomes of secondary reproductive females (nymphoid neotenics). Thirteen transcriptomes were used: 10 of Reticulitermes flavipes and R. grassei sequenced from a previous study, and two transcriptomes of R. lucifugus sequenced for the present study. After transcriptome assembly and read mapping, we examined interspecific variations of genes expressed by termites or gut microorganisms. A total of 18,323 orthologous gene clusters were detected. Functional annotation and taxonomic assignment were performed on a total of 41,287 predicted contigs in the three termite species. Between the termite species studied, functional categories of genes were comparable. Gene ontology (GO) terms analysis allowed the discovery of 9 cellulases and a total of 79 contigs potentially involved in 11 enzymatic activities used in wood metabolism. Altogether, results of this study illustrate the strong potential for the use of comparative interspecific transcriptomes, representing a complete resource for future studies including differentially expressed genes between castes or SNP analysis for population genetics. PMID:26698123

RNA sequencing approaches to transcriptomeanalysis require a large amount of input total RNA to yield sufficient mRNA using either poly-A selection or depletion of rRNA. This feature makes it difficult to miniaturize transcriptomeanalysis for greater efficiency. To address this challenge, we devised and validated a simple procedure for the preparation of whole-transcriptome cDNA libraries from a minute amount (500 pg) of total RNA. We compared a single-sample library prepared by this Ovation® RNA-Seq system with two available methods of mRNA enrichment (TruSeq™ poly-A enrichment and RiboMinus™ rRNA depletion). Using the Ovation® preparation method for a set of eight mouse tissue samples, the RNA sequencing data obtained from two different next-generation sequencing platforms (SOLiD and Illumina Genome Analyzer IIx) yielded negligible rRNA reads (<3.5%) while retaining transcriptome sequencing fidelity. We further validated the Ovation® amplification technique by examining the resulting library complexity, reproducibility, evenness of transcript coverage, 5′ and 3′ bias and platform-specific biases. Notably, in this side-by-side comparison, SOLiD sequencing chemistry is biased toward higher GC content of transcriptome and Illumina Genome analyzer IIx is biased away from neutral to lower GC content of the transcriptomics regions. PMID:21737426

Termites are eusocial insects related to cockroaches that feed on lignocellulose. These insects are key species in ecosystems since they recycle a large amount of nutrients but also are pests, exerting major economic impacts. Knowledge on the molecular pathways underlying reproduction, caste differentiation or lignocellulose digestion would largely benefit from additional transcriptomic data. This study focused on transcriptomes of secondary reproductive females (nymphoid neotenics). Thirteen transcriptomes were used: 10 of Reticulitermes flavipes and R. grassei sequenced from a previous study, and two transcriptomes of R. lucifugus sequenced for the present study. After transcriptome assembly and read mapping, we examined interspecific variations of genes expressed by termites or gut microorganisms. A total of 18,323 orthologous gene clusters were detected. Functional annotation and taxonomic assignment were performed on a total of 41,287 predicted contigs in the three termite species. Between the termite species studied, functional categories of genes were comparable. Gene ontology (GO) terms analysis allowed the discovery of 9 cellulases and a total of 79 contigs potentially involved in 11 enzymatic activities used in wood metabolism. Altogether, results of this study illustrate the strong potential for the use of comparative interspecific transcriptomes, representing a complete resource for future studies including differentially expressed genes between castes or SNP analysis for population genetics. PMID:26698123

The large-scale loach (Paramisgurnus dabryanus, Cypriniformes) is a bottom-dwelling freshwater species of fish found mainly in eastern Asia. The natural germplasm resources of this important aquaculture species has been recently threatened due to overfishing and artificial propagation. The objective of this study is to obtain the first functional genomic resource and candidate molecular markers for future conservation and breeding research. Illumina paired-end sequencing generated over one hundred million reads that resulted in 71,887 assembled transcripts, with an average length of 1465bp. 42,093 (58.56%) protein-coding sequences were predicted; and 43,837 transcripts had significant matches to NCBI nonredundant protein (Nr) database. 29,389 and 14,419 transcripts were assigned into gene ontology (GO) categories and Eukaryotic Orthologous Groups (KOG), respectively. 22,102 (31.14%) transcripts were mapped to 302 KEGG pathways. In addition, 15,106 candidate SSR markers were identified, with 11,037 pairs of PCR primers designed. 400 primers pairs of SSR selected randomly were validated, of which 364 (91%) pairs of primers were able to produce PCR products. Further test with 41 loci and 20 large-scale loach specimens collected from the four largest lakes in China showed that 36 (87.8%) loci were polymorphic. The transcriptomic profile and SSR repertoire obtained in this study will facilitate population genetic studies and selective breeding of large-scale loach in the future. PMID:25528212

Background Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing yet an untapped source for industrial applications. Results We used next generation sequencing technology to examine the transcriptome of turion development triggered by exogenous ABA. A total of 208 genes showed more than a 4-fold increase compared with 154 down-regulated genes in developing turions. The analysis of up-regulated differential expressed genes in response to dormancy exposed an enriched interplay among various pathways: signal transduction, seed dehydration, carbohydrate and secondary metabolism, and senescence. On the other side, the genes responsible for rapid growth and biomass accumulation through DNA assembly, protein synthesis and carbon fixation are repressed. Noticeably, three members of late embryogenesis abundant protein family are exclusively expressed during turion formation. High expression level of key genes in starch synthesis are APS1, APL3 and GBSSI, which could artificially be reduced for re-directing carbon flow from photosynthesis to create a higher energy biomass. Conclusions The identification and functional annotation of differentially expressed genes open a major step towards understanding the molecular network underlying vegetative frond dormancy. Moreover, genes have been identified that could be engineered in duckweeds for practical applications easing agricultural production of food crops. PMID:24456086

RNA sequencing (RNA-seq) has revolutionary roles in transcriptome identification and quantification of different types of tissues and cells in many organisms. Although numerous RNA-seq data derived from many types of human tissues and cell lines, little is known on the transcriptome repertoire of human aortic valve. In this study, we sequenced the total RNA prepared from two calcified human aortic valves and reported the whole transcriptome of human aortic valve. Integrating RNA-seq data of 13 human tissues from Human Body Map 2 Project, we constructed a transcriptome repertoire of human tissues, including 19,505 protein-coding genes and 4,948 long intergenic noncoding RNAs (lincRNAs). Among them, 263 lincRNAs were identified as novel noncoding transcripts in our data. By comparing transcriptome data among different human tissues, we observed substantial tissue specificity of RNA transcripts, both protein-coding genes and lincRNAs, in human aortic valve. Further analysis revealed that aortic valve-specific lincRNAs were more likely to be recently derived from repetitive elements in the primate lineage, but were less likely to be conserved at the nucleotide level. Expression profiling analysis showed significant lower expression levels of aortic valve-specific protein-coding genes and lincRNA genes, when compared with genes that were universally expressed in various tissues. Isoform-level expression analysis also showed that a majority of mRNA genes had a major isoform expressed in the human aortic valve. To our knowledge, this is the first comparative transcriptomeanalysis between human aortic valve and other human tissues. Our results are helpful to understand the transcriptome diversity of human tissues and the underlying mechanisms that drive tissue specificity of protein-coding genes and lincRNAs in human aortic valve. PMID:27493474

RNA sequencing (RNA-seq) has revolutionary roles in transcriptome identification and quantification of different types of tissues and cells in many organisms. Although numerous RNA-seq data derived from many types of human tissues and cell lines, little is known on the transcriptome repertoire of human aortic valve. In this study, we sequenced the total RNA prepared from two calcified human aortic valves and reported the whole transcriptome of human aortic valve. Integrating RNA-seq data of 13 human tissues from Human Body Map 2 Project, we constructed a transcriptome repertoire of human tissues, including 19,505 protein-coding genes and 4,948 long intergenic noncoding RNAs (lincRNAs). Among them, 263 lincRNAs were identified as novel noncoding transcripts in our data. By comparing transcriptome data among different human tissues, we observed substantial tissue specificity of RNA transcripts, both protein-coding genes and lincRNAs, in human aortic valve. Further analysis revealed that aortic valve-specific lincRNAs were more likely to be recently derived from repetitive elements in the primate lineage, but were less likely to be conserved at the nucleotide level. Expression profiling analysis showed significant lower expression levels of aortic valve-specific protein-coding genes and lincRNA genes, when compared with genes that were universally expressed in various tissues. Isoform-level expression analysis also showed that a majority of mRNA genes had a major isoform expressed in the human aortic valve. To our knowledge, this is the first comparative transcriptomeanalysis between human aortic valve and other human tissues. Our results are helpful to understand the transcriptome diversity of human tissues and the underlying mechanisms that drive tissue specificity of protein-coding genes and lincRNAs in human aortic valve. PMID:27493474

As the conduit for nutrients and growth signals, the placenta is critical to establishing an environment sufficient for fetal growth and development. To better understand the mechanisms regulating placental development and gene expression, we characterized the transcriptome of term placenta from 20 ...

The embryonic transcriptome of the Caribbean fruit fly, Anastrepha suspensa, was sequenced by 454 pyrosequencing in an effort to isolate embryonic promoters and genes involved in programmed cell death. A cDNA library was constructed from total RNA pooled from various time points in embryogenesis usi...

The evolution of specialized tissues with novel functions, such as the silk synthesizing glands in spiders, is likely an influential driver of adaptive success. Large-scale gene duplication events and subsequent paralog divergence are thought to be required for generating evolutionary novelty. Such an event has been proposed for spiders, but not tested. We de novo assembled transcriptomes from three cobweb weaving spider species. Based on phylogenetic analyses of gene families with representatives from each of the three species, we found numerous duplication events indicative of a whole genome or segmental duplication. We estimated the age of the gene duplications relative to several speciation events within spiders and arachnids and found that the duplications likely occurred after the divergence of scorpions (order Scorpionida) and spiders (order Araneae), but before the divergence of the spider suborders Mygalomorphae and Araneomorphae, near the evolutionary origin of spider silk glands. Transcripts that are expressed exclusively or primarily within black widow silk glands are more likely to have a paralog descended from the ancient duplication event and have elevated amino acid replacement rates compared with other transcripts. Thus, an ancient large-scale gene duplication event within the spider lineage was likely an important source of molecular novelty during the evolution of silk gland-specific expression. This duplication event may have provided genetic material for subsequent silk gland diversification in the true spiders (Araneomorphae). PMID:26058392

The evolution of specialized tissues with novel functions, such as the silk synthesizing glands in spiders, is likely an influential driver of adaptive success. Large-scale gene duplication events and subsequent paralog divergence are thought to be required for generating evolutionary novelty. Such an event has been proposed for spiders, but not tested. We de novo assembled transcriptomes from three cobweb weaving spider species. Based on phylogenetic analyses of gene families with representatives from each of the three species, we found numerous duplication events indicative of a whole genome or segmental duplication. We estimated the age of the gene duplications relative to several speciation events within spiders and arachnids and found that the duplications likely occurred after the divergence of scorpions (order Scorpionida) and spiders (order Araneae), but before the divergence of the spider suborders Mygalomorphae and Araneomorphae, near the evolutionary origin of spider silk glands. Transcripts that are expressed exclusively or primarily within black widow silk glands are more likely to have a paralog descended from the ancient duplication event and have elevated amino acid replacement rates compared with other transcripts. Thus, an ancient large-scale gene duplication event within the spider lineage was likely an important source of molecular novelty during the evolution of silk gland-specific expression. This duplication event may have provided genetic material for subsequent silk gland diversification in the true spiders (Araneomorphae). PMID:26058392

Leaves are vital organs for biomass and seed production because of their role in the generation of metabolic energy and organic compounds. A better understanding of the molecular networks underlying leaf development is crucial to sustain global requirements for food and renewable energy. Here, we combined transcriptome profiling of proliferative leaf tissue with in-depth phenotyping of the fourth leaf at later stages of development in 197 recombinant inbred lines of two different maize (Zea mays) populations. Previously, correlation analysis in a classical biparental mapping population identified 1,740 genes correlated with at least one of 14 traits. Here, we extended these results with data from a multiparent advanced generation intercross population. As expected, the phenotypic variability was found to be larger in the latter population than in the biparental population, although general conclusions on the correlations among the traits are comparable. Data integration from the two diverse populations allowed us to identify a set of 226 genes that are robustly associated with diverse leaf traits. This set of genes is enriched for transcriptional regulators and genes involved in protein synthesis and cell wall metabolism. In order to investigate the molecular network context of the candidate gene set, we integrated our data with publicly available functional genomics data and identified a growth regulatory network of 185 genes. Our results illustrate the power of combining in-depth phenotyping with transcriptomics in mapping populations to dissect the genetic control of complex traits and present a set of candidate genes for use in biomass improvement. PMID:26754667

Transcriptome sequencing is now widely adopted as an efficient means to study the chemical diversity of venoms. To improve the efficiency of analysis of these large datasets, we have optimised an analysis pipeline for cone snail venom gland transcriptomes. The pipeline combines ConoSorter with sequence architecture-based elimination and similarity searching using BLAST to improve the accuracy of sequence identification and classification, while reducing requirements for manual intervention. As a proof-of-concept, we used this approach reanalysed three previously published cone snail transcriptomes from diverse dietary groups. Our pipeline method generated similar results to the published studies with significantly less manual intervention. We additionally found undiscovered sequences in the piscovorous Conus geographus and vermivorous Conus miles and identified sequences in incorrect superfamilies in the molluscivorus Conus marmoreus and C. geographus transcriptomes. Our results indicate that this method can improve toxin detection without extending analysis time. While this method was evaluated on cone snail transcriptomes it can be easily optimised to retrieve toxins from other venomous animals. PMID:26376071

Genome-wide analyses and high-throughput screening was long reserved for biomedical applications and genetic model organisms. With the rapid development of massively parallel sequencing nanotechnology (or next-generation sequencing) and simultaneous maturation of bioinformatic tools, this situation has dramatically changed. Genome-wide thinking is forging its way into disciplines like evolutionary biology or molecular ecology that were historically confined to small-scale genetic approaches. Accessibility to genome-scale information is transforming these fields, as it allows us to answer long-standing questions like the genetic basis of local adaptation and speciation or the evolution of gene expression profiles that until recently were out of reach. Many in the eco-evolutionary sciences will be working with large-scale genomic data sets, and a basic understanding of the concepts and underlying methods is necessary to judge the work of others. Here, I briefly introduce next-generation sequencing and then focus on transcriptome shotgun sequencing (RNA-seq). This article gives a broad overview and provides practical guidance for the many steps involved in a typical RNA-seq work flow from sampling, to RNA extraction, library preparation and data analysis. I focus on principles, present useful tools where appropriate and point out where caution is needed or progress to be expected. This tutorial is mostly targeted at beginners, but also contains potentially useful reflections for the more experienced. PMID:23621713

Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics. PMID:27365449

Enterococcus faecalis is the most commonly isolated species from endodontic failure root canals; its persistence in treated root canals has been attributed to its ability to resist high pH stress. The goal of this study was to characterize the E. faecalis transcriptome and to identify candidate genes for response and resistance to alkaline stress using Illumina HiSeq 2000 sequencing. We found that E. faecalis could survive and form biofilms in a pH 10 environment and that alkaline stress had a great impact on the transcription of many genes in the E. faecalis genome. The transcriptome sequencing results revealed that 613 genes were differentially expressed (DEGs) for E. faecalis grown in pH 10 medium; 211 genes were found to be differentially up-regulated and 402 genes differentially down-regulated. Many of the down-regulated genes found are involved in cell energy production and metabolism and carbohydrate and amino acid metabolism, and the up-regulated genes are mostly related to nucleotide transport and metabolism. The results presented here reveal that cultivation of E. faecalis in alkaline stress has a profound impact on its transcriptome. The observed regulation of genes and pathways revealed that E. faecalis reduced its carbohydrate and amino acid metabolism and increased nucleotide synthesis to adapt and grow in alkaline stress. A number of the regulated genes may be useful candidates for the development of new therapeutic approaches for the treatment of E. faecalis infections. PMID:26300863

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation. PMID:27032112

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation. PMID:27032112

The placenta is the essential organ of mammalian pregnancy and errors in its development and function are associated with a wide range of human pathologies of pregnancy. Genome sequencing has led to methods for investigation of the transcriptome (all expressed RNA species) using microarrays and next-generation sequencing, and implementation of these techniques has identified many novel species of RNA including: micro-RNA, long noncoding RNA, and circular RNA. These species can physically interact with both each other and regulatory proteins to modify gene expression and messenger RNA to protein translation. Transcriptomeanalysis is actively used to investigate placental development and dysfunction in pathologies ranging from preeclampsia and fetal growth restriction to preterm labor. Genome-wide gene expression analysis is also being applied to identify prognostic and diagnostic biomarkers of these disorders. In this comprehensive review we summarize transcriptome biology, methods of isolation and analysis, application to placental development and pathology, and use in diagnostic analysis in maternal blood. Key information for analysis methods is organized into quick reference tables where current analysis techniques and tools are cited and compared. We have created this review as a practical guide and starting reference for those interested in beginning an investigation into the transcriptome of the placenta. PMID:26428493

The grape and wine industries are heavily reliant on sulphite preservatives. However, the view that sulphites act directly on bacterial and fungal pathogens may be simplistic. Mechanisms of sulphur-enhanced defences are largely unknown; many sulphur-rich compounds enhance plant defences and sulphite can also have oxidative consequences via production of H(2)O(2) or sulphitolysis. To investigate the effects of sulphur dioxide (SO(2) ) on fresh table grapes (Vitis vinifera L. 'Crimson Seedless'), transcriptomeanalysis was carried out on berries treated with SO(2) under commercial conditions for 21 d. We found a broad perturbation of metabolic processes, consistent with a large-scale stress response. Transcripts encoding putative sulphur-metabolizing enzymes indicated that sulphite was directed towards chelation and conjugation, and away from oxidation to sulphate. The results indicated that redox poise was altered dramatically by SO(2) treatment, evidenced by alterations in plastid and mitochondrial alternative electron transfer pathways, up-regulation of fermentation transcripts and numerous glutathione S-transferases, along with a down-regulation of components involved in redox homeostasis. Features of biotic stress were up-regulated, notably signalling via auxin, ethylene and jasmonates. Taken together, this inventory of transcriptional responses is consistent with a long-term cellular response to oxidative stress, similar to the effects of reactive oxygen species. PMID:21689113

Monochromatic light is widely applied to promote poultry reproductive performance, yet little is currently known regarding the mechanism by which light wavelengths affect pigeon reproduction. Recently, high-throughput sequencing technologies have been used to provide genomic information for solving this problem. In this study, we employed Illumina Hiseq 2000 to identify differentially expressed genes in ovary tissue from pigeons under blue and white light conditions and de novo transcriptome assembly to construct a comprehensive sequence database containing information on the mechanisms of follicle development. A total of 157,774 unigenes (mean length: 790 bp) were obtained by the Trinity program, and 35.83% of these unigenes were matched to genes in a non-redundant protein database. Gene description, gene ontology, and the clustering of orthologous group terms were performed to annotate the transcriptome assembly. Differentially expressed genes between blue and white light conditions included those related to oocyte maturation, hormone biosynthesis, and circadian rhythm. Furthermore, 17,574 SSRs and 533,887 potential SNPs were identified in this transcriptome assembly. This work is the first transcriptomeanalysis of the Columba ovary using Illumina technology, and the resulting transcriptome and differentially expressed gene data can facilitate further investigations into the molecular mechanism of the effect of blue light on follicle development and reproduction in pigeons and other bird species. PMID:26599806

A comprehensive transcriptomeanalysis has been performed on protein-coding RNAs of Strongylocentrotus purpuratus, including 10 different embryonic stages, six feeding larval and metamorphosed juvenile stages, and six adult tissues. In this study, we pooled the transcriptomes from all of these sources and focused on the insights they provide for gene structure in the genome of this recently sequenced model system. The genome had initially been annotated by use of computational gene model prediction algorithms. A large fraction of these predicted genes were recovered in the transcriptome when the reads were mapped to the genome and appropriately filtered and analyzed. However, in a manually curated subset, we discovered that more than half the computational gene model predictions were imperfect, containing errors such as missing exons, prediction of nonexistent exons, erroneous intron/exon boundaries, fusion of adjacent genes, and prediction of multiple genes from single genes. The transcriptome data have been used to provide a systematic upgrade of the gene model predictions throughout the genome, very greatly improving the research usability of the genomic sequence. We have constructed new public databases that incorporate information from the transcriptome analyses. The transcript-based gene model data were used to define average structural parameters for S. purpuratus protein-coding genes. In addition, we constructed a custom sea urchin gene ontology, and assigned about 7000 different annotated transcripts to 24 functional classes. Strong correlations became evident between given functional ontology classes and structural properties, including gene size, exon number, and exon and intron size. PMID:22709795

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes. PMID:25060758

Machine learning (ML) is an intelligent data mining technique that builds a prediction model based on the learning of prior knowledge to recognize patterns in large-scale data sets. We present an ML-based methodology for transcriptomeanalysis via comparison of gene coexpression networks, implemented as an R package called machine learning-based differential network analysis (mlDNA) and apply this method to reanalyze a set of abiotic stress expression data in Arabidopsis thaliana. The mlDNA first used a ML-based filtering process to remove nonexpressed, constitutively expressed, or non-stress-responsive "noninformative" genes prior to network construction, through learning the patterns of 32 expression characteristics of known stress-related genes. The retained "informative" genes were subsequently analyzed by ML-based network comparison to predict candidate stress-related genes showing expression and network differences between control and stress networks, based on 33 network topological characteristics. Comparative evaluation of the network-centric and gene-centric analytic methods showed that mlDNA substantially outperformed traditional statistical testing-based differential expression analysis at identifying stress-related genes, with markedly improved prediction accuracy. To experimentally validate the mlDNA predictions, we selected 89 candidates out of the 1784 predicted salt stress-related genes with available SALK T-DNA mutagenesis lines for phenotypic screening and identified two previously unreported genes, mutants of which showed salt-sensitive phenotypes. PMID:24520154

The human liver plays a vital role in meeting the body's metabolic needs and maintaining homeostasis. To address the molecular mechanisms of liver function, we integrated multiple gene expression datasets from microarray, MPSS, SAGE and EST platforms to generate a transcriptome atlas of the normal human liver. Our results show that 17396 genes are expressed in the human liver. 238 genes were identified as liver enrichment genes, involved in the functions of immune response and metabolic processes, from the MPSS and EST datasets. A comparative analysis of liver transcriptomes was performed in humans, mice and rats with microarray datasets shows that the expression profile of homologous genes remains significantly different between mouse/rat and human, suggesting a functional variance and regulation bias of genes expressed in the livers. The integrated liver transcriptome data should provide a valuable resource for the in-depth understanding of human liver biology and liver disease. PMID:20800674

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are liver originated malignant tumors. Of the two, ICC has the worse prognosis because it has no reliable diagnostic markers and its carcinogenic mechanism is not fully understood. The aim of this study was to integrate metabolomics and transcriptomics datasets to identify variances if any in the carcinogenic mechanism of ICC and HCC. Ten ICC and 6 HCC who were resected surgically, were enrolled. miRNA and mRNA expression analysis were performed by microarray on ICC and HCC and their corresponding non-tumor tissues (ICC_NT and HCC_NT). Compound analysis was performed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Principle component analysis (PCA) revealed that among the four sample groups (ICC, ICC_NT, HCC, and HCC_NT) there were 14 compounds, 62 mRNAs and 17 miRNAs with two distinct patterns: tumor and non-tumor, and ICC and non-ICC. We accurately (84.38%) distinguished ICC by the distinct pattern of its compounds. Pathway analysis using transcriptome and metabolome showed that several pathways varied between tumor and non-tumor samples. Based on the results of the PCA, we believe that ICC and HCC have different carcinogenic mechanism therefore knowing the specific profile of genes and compounds can be useful in diagnosing ICC. PMID:26538415

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are liver originated malignant tumors. Of the two, ICC has the worse prognosis because it has no reliable diagnostic markers and its carcinogenic mechanism is not fully understood. The aim of this study was to integrate metabolomics and transcriptomics datasets to identify variances if any in the carcinogenic mechanism of ICC and HCC. Ten ICC and 6 HCC who were resected surgically, were enrolled. miRNA and mRNA expression analysis were performed by microarray on ICC and HCC and their corresponding non-tumor tissues (ICC_NT and HCC_NT). Compound analysis was performed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Principle component analysis (PCA) revealed that among the four sample groups (ICC, ICC_NT, HCC, and HCC_NT) there were 14 compounds, 62 mRNAs and 17 miRNAs with two distinct patterns: tumor and non-tumor, and ICC and non-ICC. We accurately (84.38%) distinguished ICC by the distinct pattern of its compounds. Pathway analysis using transcriptome and metabolome showed that several pathways varied between tumor and non-tumor samples. Based on the results of the PCA, we believe that ICC and HCC have different carcinogenic mechanism therefore knowing the specific profile of genes and compounds can be useful in diagnosing ICC. PMID:26538415

Myceliophthora thermophila is a thermophilic fungus whose genome encodes a wide range of carbohydrate-active enzymes (CAZymes) involved in plant biomass degradation. Such enzymes have potential applications in turning different kinds of lignocellulosic feedstock into sugar precursors for biofuels and chemicals. The present study examined and compared the transcriptomes and exoproteomes of M. thermophila during cultivation on different types of complex biomass to gain insight into how its secreted enzymatic machinery varies with different sources of lignocellulose. In the transcriptomeanalysis three monocot (barley, oat, triticale) and three dicot (alfalfa, canola, flax) plants were used whereas in the proteome analysis additional substrates, i.e. wood and corn stover pulps, were included. A core set of 59 genes encoding CAZymes was up-regulated in response to both monocot and dicot straws, including nine polysaccharide monooxygenases and GH10, but not GH11, xylanases. Genes encoding additional xylanolytic enzymes were up-regulated during growth on monocot straws, while genes encoding additional pectinolytic enzymes were up-regulated in response to dicot biomass. Exoproteome analysis was generally consistent with the conclusions drawn from transcriptomeanalysis, but additional CAZymes that accumulated to high levels were identified. Despite the wide variety of biomass sources tested some CAZy family members were not expressed under any condition. The results of this study provide a comprehensive view from both transcriptome and exoproteome levels, of how M. thermophila responds to a wide range of biomass sources using its genomic resources. PMID:24881579

Seedlessness is desirable for most citrus fruit, and identification of spontaneous or irradiated seedless mutants is important in developing citrus cultivars. We conducted a transcriptomeanalysis in early fruit development of three seedy citrus types (‘Fallglo’, a largely C. reticulata hybrid; ‘Pi...

Methylation of DNA molecules is a key mechanism associated with human disease, altered gene expression and phenotype. Using reduced representation bisulphite sequencing (RRBS) technology we have analysed DNA methylation patterns in healthy individuals and identified genes showing significant inter-individual variation. Further, using whole genome transcriptomeanalysis (RNA-Seq) on the same individuals we showed a local and specific relationship of exon inclusion and variable DNA methylation pattern. For RRBS, 363 million, 100-bp reads were generated from 13 samples using Illumina GAII and HiSeq2000 platforms. Here we also present additional RRBS data for a female pair of monozygotic twins that was not described in our original publication. Further, We performed RNA-Seq on four of these individuals, generating 174 million, 51-bp high quality reads on an Illumina HiSeq2000 platform. The current data set could be exploited as a comprehensive resource for understanding the nature and mechanism of variable phenotypic traits and altered disease susceptibility due to variable DNA methylation and gene expression patterns in healthy individuals. PMID:26978482

Methylation of DNA molecules is a key mechanism associated with human disease, altered gene expression and phenotype. Using reduced representation bisulphite sequencing (RRBS) technology we have analysed DNA methylation patterns in healthy individuals and identified genes showing significant inter-individual variation. Further, using whole genome transcriptomeanalysis (RNA-Seq) on the same individuals we showed a local and specific relationship of exon inclusion and variable DNA methylation pattern. For RRBS, 363 million, 100-bp reads were generated from 13 samples using Illumina GAII and HiSeq2000 platforms. Here we also present additional RRBS data for a female pair of monozygotic twins that was not described in our original publication. Further, We performed RNA-Seq on four of these individuals, generating 174 million, 51-bp high quality reads on an Illumina HiSeq2000 platform. The current data set could be exploited as a comprehensive resource for understanding the nature and mechanism of variable phenotypic traits and altered disease susceptibility due to variable DNA methylation and gene expression patterns in healthy individuals. PMID:26978482

The insectivorous Venus flytrap (Dionaea muscipula) is renowned from Darwin’s studies of plant carnivory and the origins of species. To provide tools to analyze the evolution and functional genomics of D. muscipula, we sequenced a normalized cDNA library synthesized from mRNA isolated from D. muscipula flowers and traps. Using the Oases transcriptome assembler 79,165,657 quality trimmed reads were assembled into 80,806 cDNA contigs, with an average length of 679 bp and an N50 length of 1,051 bp. A total of 17,047 unique proteins were identified, and assigned to Gene Ontology (GO) and classified into functional categories. A total of 15,547 full-length cDNA sequences were identified, from which open reading frames were detected in 10,941. Comparative GO analyses revealed that D. muscipula is highly represented in molecular functions related to catalytic, antioxidant, and electron carrier activities. Also, using a single copy sequence PCR-based method, we estimated that the genome size of D. muscipula is approx. 3 Gb. Our genome size estimate and transcriptome analyses will contribute to future research on this fascinating, monotypic species and its heterotrophic adaptations. PMID:25886597

The insectivorous Venus flytrap (Dionaea muscipula) is renowned from Darwin's studies of plant carnivory and the origins of species. To provide tools to analyze the evolution and functional genomics of D. muscipula, we sequenced a normalized cDNA library synthesized from mRNA isolated from D. muscipula flowers and traps. Using the Oases transcriptome assembler 79,165,657 quality trimmed reads were assembled into 80,806 cDNA contigs, with an average length of 679 bp and an N50 length of 1,051 bp. A total of 17,047 unique proteins were identified, and assigned to Gene Ontology (GO) and classified into functional categories. A total of 15,547 full-length cDNA sequences were identified, from which open reading frames were detected in 10,941. Comparative GO analyses revealed that D. muscipula is highly represented in molecular functions related to catalytic, antioxidant, and electron carrier activities. Also, using a single copy sequence PCR-based method, we estimated that the genome size of D. muscipula is approx. 3 Gb. Our genome size estimate and transcriptome analyses will contribute to future research on this fascinating, monotypic species and its heterotrophic adaptations. PMID:25886597

Spider venoms are complex cocktails rich in peptides, proteins and organic molecules that collectively act to immobilize prey. Venoms of the primitive hunting spider, Plectreurys tristis, have numerous neurotoxic peptides called “plectoxins” (PLTX), a unique acylpolyamine called bis(agmatine)oxalamide, and larger unidentified protein components. These spiders also have unconventional multi-lobed venom glands. Inspired by these unusual characteristics and their phylogenetic position as Haplogynes, we have partially characterized the venome of P. tristis using combined transcriptomic and proteomic methods. With these analyses we found known venom neurotoxins U1-PLTX-Pt1a, U3-PLTX-Pt1a, and we discovered new groups of potential neurotoxins, expanding the U1- and ω-PLTX families and adding U4-through U9-PLTX as six new groups. The venom also contains proteins that are homologs of astacin metalloproteases that, combined with venom peptides, make up 94% of components detected in crude venom, while the remaining 6% is a single undescribed protein with unknown function. Other proteins detected in the transcriptome were found to be members of conserved gene families and make up 20% of the transcripts. These include cDNA sequences that match venom proteins from Mesobuthus and Hottentotta scorpions, Loxosceles and Dysdera spiders, and also salivary and secreted peptide sequences from Ixodes, Amblyomma and Rhipicephalus ticks. Finally, we show that crude venom has neurotoxic effects and an effective paralytic dose on crickets of 3.3µg/gm. PMID:25400903

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters. PMID:25164755

Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, Anthonomus grandis, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, A. grandis, was analyzed. The A. grandis transcriptomeanalysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to Tribolium castaneum sequences. A comparison of A. grandis, Drosophila melanogaster and Bombyx mori protein families’ data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with T. castaneum SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to A. grandis female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, A. grandis. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects. PMID:24386449

Background The diamondback moth (DBM, Plutella xylostella) is a crucifer-specific pest that causes significant crop losses worldwide. Barbarea vulgaris (Brassicaceae) can resist DBM and other herbivorous insects by producing feeding-deterrent triterpenoid saponins. Plant breeders have long aimed to transfer this insect resistance to other crops. However, a lack of knowledge on the biosynthetic pathways and regulatory networks of these insecticidal saponins has hindered their practical application. A pyrosequencing-based transcriptomeanalysis of B. vulgaris during DBM larval feeding was performed to identify genes and gene networks responsible for saponin biosynthesis and its regulation at the genome level. Principal Findings Approximately 1.22, 1.19, 1.16, 1.23, 1.16, 1.20, and 2.39 giga base pairs of clean nucleotides were generated from B. vulgaris transcriptomes sampled 1, 4, 8, 12, 24, and 48 h after onset of P. xylostella feeding and from non-inoculated controls, respectively. De novo assembly using all data of the seven transcriptomes generated 39,531 unigenes. A total of 37,780 (95.57%) unigenes were annotated, 14,399 of which were assigned to one or more gene ontology terms and 19,620 of which were assigned to 126 known pathways. Expression profiles revealed 2,016–4,685 up-regulated and 557–5188 down-regulated transcripts. Secondary metabolic pathways, such as those of terpenoids, glucosinolates, and phenylpropanoids, and its related regulators were elevated. Candidate genes for the triterpene saponin pathway were found in the transcriptome. Orthological analysis of the transcriptome with four other crucifer transcriptomes identified 592 B. vulgaris-specific gene families with a P-value cutoff of 1e−5. Conclusion This study presents the first comprehensive transcriptomeanalysis of B. vulgaris subjected to a series of DBM feedings. The biosynthetic and regulatory pathways of triterpenoid saponins and other DBM deterrent metabolites in this plant were

The molecular mechanisms that drive the development of the endangered fossil fish species Acipenser baeri are difficult to study due to the lack of genomic data. Recent advances in sequencing technologies and the reducing cost of sequencing offer exclusive opportunities for exploring important molecular mechanisms underlying specific biological processes. This manuscript describes the large scale sequencing and analyses of mRNA from Acipenser baeri collected at five development time points using the Illumina Hiseq2000 platform. The sequencing reads were de novo assembled and clustered into 278167 unigenes, of which 57346 (20.62%) had 45837 known homologues proteins in Uniprot protein databases while 11509 proteins matched with at least one sequence of assembled unigenes. The remaining 79.38% of unigenes could stand for non-coding unigenes or unigenes specific to A. baeri. A number of 43062 unigenes were annotated into functional categories via Gene Ontology (GO) annotation whereas 29526 unigenes were associated with 329 pathways by mapping to KEGG database. Subsequently, 3479 differentially expressed genes were scanned within developmental stages and clustered into 50 gene expression profiles. Genes preferentially expressed at each stage were also identified. Through GO and KEGG pathway enrichment analysis, relevant physiological variations during the early development of A. baeri could be better cognized. Accordingly, the present study gives insights into the transcriptome profile of the early development of A. baeri, and the information contained in this large scaletranscriptome will provide substantial references for A. baeri developmental biology and promote its aquaculture research. PMID:26359664

Alveolar macrophages (AMs) reside on the luminal surfaces of the airways and alveoli where they maintain host defense and promote alveolar homeostasis by ingesting inhaled particulates and regulating inflammatory responses. Recent studies have demonstrated that AMs populate the lungs during embryogenesis and self-renew throughout life with minimal replacement by circulating monocytes, except under extreme conditions of depletion or radiation injury. Here we demonstrate that on a global scale, environment appears to dictate AM development and function. Indeed, transcriptomeanalysis of embryonic host-derived and postnatal donor-derived AMs coexisting within the same mouse demonstrated >98% correlation and overall functional analyses were similar. However, we also identified several genes whose expression was dictated by origin rather than environment. The most differentially expressed gene not altered by environment was Marco, a gene recently demonstrated to have enhancer activity in embryonic-derived but not postnatal-derived tissue macrophages. Overall, we show that under homeostatic conditions, the environment largely dictates the programming and function of AMs, whereas the expression of a small number of genes remains linked to the origin of the cell. PMID:26232173

Pseudoperonospora cubensis, an oomycete, is the causal agent of cucurbit downy mildew, and is responsible for significant losses on cucurbit crops worldwide. While other oomycete plant pathogens have been extensively studied at the molecular level, Ps. cubensis and the molecular basis of its interaction with cucurbit hosts has not been well examined. Here, we present the first large-scale global gene expression analysis of Ps. cubensis infection of a susceptible Cucumis sativus cultivar, 'Vlaspik', and identification of genes with putative roles in infection, growth, and pathogenicity. Using high throughput whole transcriptome sequencing, we captured differential expression of 2383 Ps. cubensis genes in sporangia and at 1, 2, 3, 4, 6, and 8 days post-inoculation (dpi). Additionally, comparison of Ps. cubensis expression profiles with expression profiles from an infection time course of the oomycete pathogen Phytophthora infestans on Solanum tuberosum revealed similarities in expression patterns of 1,576-6,806 orthologous genes suggesting a substantial degree of overlap in molecular events in virulence between the biotrophic Ps. cubensis and the hemi-biotrophic P. infestans. Co-expression analyses identified distinct modules of Ps. cubensis genes that were representative of early, intermediate, and late infection stages. Collectively, these expression data have advanced our understanding of key molecular and genetic events in the virulence of Ps. cubensis and thus, provides a foundation for identifying mechanism(s) by which to engineer or effect resistance in the host. PMID:22545137

Background The oriental river prawn (Macrobrachium nipponense) is the most prevalent aquaculture species in China. The sexual precocity in this species has received considerable attention in recent years because more and more individuals matured at a small size, which devalues the commercial production. In this study, we developed deep-coverage transcriptomic sequencing data for the ovaries of sexually precocious and normal sexually mature M. nipponense using next-generation RNA sequencing technology and attempted to provide the first insight into the molecular regulatory mechanism of sexual precocity in this species. Results A total of 63,336 unigenes were produced from the ovarian cDNA libraries of sexually precocious and normal sexually mature M. nipponense using Illumina HiSeq 2500 platform. Through BLASTX searches against the NR, STRING, Pfam, Swissprot and KEGG databases, 15,134 unigenes were annotated, accounting for 23.89% of the total unigenes. 5,195 and 3,227 matched unigenes were categorized by GO and COG analysis respectively. 15,908 unigenes were consequently mapped into 332 KEGG pathways, and many reproduction-related pathways and genes were identified. Moreover, 26,008 SSRs were identified from 18,133 unigenes. 80,529 and 80,516 SNPs were yielded from ovarian libraries of sexually precocious and normal sexually mature prawn, respectively, and 29,851 potential SNPs between these two groups were also predicted. After comparing the ovarian libraries of sexually precocious and normal sexually mature prawn, 549 differentially expressed genes (DEGs) and 9 key DEGs that may be related to sexual precocity of M. nipponense were identified. 20 DEGs were selected for validation by quantitative real-time PCR (QPCR) and 19 DEGs show consistent expression between QPCR and RNAseq-based differential expression analysis datasets. Conclusion This is the first report on the large-scale RNA sequencing of ovaries of sexually precocious and normal sexually mature M

Host-associated differentiation is one of the driving forces behind the diversification of phytophagous insects. In this study, host induced transcriptomic differences were investigated in the sweetpotato whitefly Bemisia tabaci, an invasive agricultural pest worldwide. Comparative transcriptomic analyses using coding sequence (CDS), 5′ and 3′ untranslated regions (UTR) showed that sequence divergences between the original host plant, cabbage, and the derived hosts, including cotton, cucumber and tomato, were 0.11–0.14%, 0.19–0.26%, and 0.15–0.21%, respectively. In comparison to the derived hosts, 418 female and 303 male transcripts, respectively, were up-regulated in the original cabbage strain. Among them, 17 transcripts were consistently up-regulated in both female and male whiteflies originated from the cabbage host. Specifically, two ESTs annotated as Cathepsin B or Cathepsin B-like genes were significantly up-regulated in the original cabbage strain, representing a transcriptomic response to the dietary challenges imposed by the host shifting. Results from our transcriptomeanalysis, in conjunction with previous reports documenting the minor changes in their reproductive capacity, insecticide susceptibility, symbiotic composition and feeding behavior, suggest that the impact of host-associated differentiation in whiteflies is limited. Furthermore, it is unlikely the major factor contributing to their rapid range expansion/invasiveness. PMID:25540625

The Spanish slug, Arion vulgaris, is considered one of the hundred most invasive species in Central Europe. The immense and very successful adaptation and spreading of A. vulgaris suggest that it developed highly effective mechanisms to deal with infections and natural predators. Current transcriptomic and proteomic studies on gastropods have been restricted mainly to marine and freshwater gastropods. No transcriptomic or proteomic study on A. vulgaris has been carried out so far, and in the current study, the first transcriptomic database from adult specimen of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified; 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity including many associated pattern recognition, effector proteins and cytokine-like proteins. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs as well as for putative antimicrobial compounds for drug discovery and biomedical applications. PMID:26986963

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides. PMID:27625674

The Spanish slug, Arion vulgaris, is considered one of the hundred most invasive species in Central Europe. The immense and very successful adaptation and spreading of A. vulgaris suggest that it developed highly effective mechanisms to deal with infections and natural predators. Current transcriptomic and proteomic studies on gastropods have been restricted mainly to marine and freshwater gastropods. No transcriptomic or proteomic study on A. vulgaris has been carried out so far, and in the current study, the first transcriptomic database from adult specimen of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified; 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity including many associated pattern recognition, effector proteins and cytokine-like proteins. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs as well as for putative antimicrobial compounds for drug discovery and biomedical applications. PMID:26986963

Numerous organisms around the globe have successfully adapted to subterranean environments. A powerful system in which to study cave adaptation is the freshwater characin fish, Astyanax mexicanus. Prior studies in this system have established a genetic basis for the evolution of numerous regressive traits, most notably vision and pigmentation reduction. However, identification of the precise genetic alterations that underlie these morphological changes has been delayed by limited genetic and genomic resources. To address this, we performed a transcriptomeanalysis of cave and surface dwelling Astyanax morphs using Roche/454 pyrosequencing technology. Through this approach, we obtained 576,197 Pachón cavefish-specific reads and 438,978 surface fish-specific reads. Using this dataset, we assembled transcriptomes of cave and surface fish separately, as well as an integrated transcriptome that combined 1,499,568 reads from both morphotypes. The integrated assembly was the most successful approach, yielding 22,596 high quality contiguous sequences comprising a total transcriptome length of 21,363,556 bp. Sequence identities were obtained through exhaustive blast searches, revealing an adult transcriptome represented by highly diverse Gene Ontology (GO) terms. Our dataset facilitated rapid identification of sequence polymorphisms between morphotypes. These data, along with positional information collected from the Danio rerio genome, revealed several syntenic regions between Astyanax and Danio. We demonstrated the utility of this positional information through a QTL analysis of albinism in a surface x Pachón cave F(2) pedigree, using 65 polymorphic markers identified from our integrated assembly. We also adapted our dataset for an RNA-seq study, revealing many genes responsible for visual system maintenance in surface fish, whose expression was not detected in adult Pachón cavefish. Conversely, several metabolism-related genes expressed in cavefish were not detected

Targeted therapy is a rational and promising strategy for the treatment of advanced cancer. For the development of clinical agents targeting oncogenic signaling pathways, it is important to define the specificity of compounds to the target molecular pathway. Genome-wide transcriptomicanalysis is an unbiased approach to evaluate the compound mode of action, but it is still unknown whether the analysis could be widely applicable to classify molecularly targeted anticancer agents. We comprehensively obtained and analyzed 129 transcriptomic datasets of cancer cells treated with 83 anticancer drugs or related agents, covering most clinically used, molecularly targeted drugs alongside promising inhibitors of molecular cancer targets. Hierarchical clustering and principal component analysis revealed that compounds targeting similar target molecules or pathways were clustered together. These results confirmed that the gene signatures of these drugs reflected their modes of action. Of note, inhibitors of oncogenic kinase pathways formed a large unique cluster, showing that these agents affect a shared molecular pathway distinct from classical antitumor agents and other classes of agents. The gene signature analysis further classified kinome-targeting agents depending on their target signaling pathways, and we identified target pathway-selective signature gene sets. The gene expression analysis was also valuable in uncovering unexpected target pathways of some anticancer agents. These results indicate that comprehensive transcriptomicanalysis with our database (http://scads.jfcr.or.jp/db/cs/) is a powerful strategy to validate and re-evaluate the target pathways of anticancer compounds. PMID:25911996

Squamous lung cancer is a common type of lung cancer; however, its mechanism of oncogenesis is still unknown. The aim of this study was to screen candidate genes of squamous lung cancer using a bioinformatics strategy and elucidate the mechanism of squamous lung cancer. Published microarray data of the GSE3268 series was obtained from Gene Expression Omnibus (GEO). Significance analysis of microarrays was performed using the software R, and differentially expressed genes by R analysis were harvested. The relationship between transcription factors and target genes in cancer were collected from the Transcriptional regulatory element database. A transcriptome network analysis method was used to construct gene regulation networks and select the candidate genes for squamous lung cancer. SPI1, FLI1, FOS, ETS2, EGR1 and PPARG were defined as candidate genes for squamous lung cancer by the transcriptome network analysis method. Among them, 5 genes had been reported to be involved in lung cancer, except SPI1 and FLI1. Effective recall on previous knowledge conferred strong confidence in these methods. It is demonstrated that transcriptome network analysis is useful in the identification of candidate genes in disease. PMID:21922129

Background.Chinese fir [Cunninghamia lanceolata (Lamb.) Hook.] is one of the most important native tree species for timber production in southern China. An understanding of overall fast growing stage, stem growth stage and senescence stage cambium transcriptome variation is lacking. We used transcriptome sequencing to identify the repertoire of genes expressed during development of xylem tissue in Chinese fir, aiming to delineate the molecular mechanisms of wood formation. Results. We carried out transcriptome sequencing at three different cultivation ages (7Y, 15Y and 21Y) generating 68.71 million reads (13.88 Gbp). A total of 140,486 unigenes with a mean size of 568.64 base pairs (bp) were obtained via de novo assembly. Of these, 27,427 unigenes (19.52%) were further annotated by comparison to public protein databases. A total of 5,331 (3.79%) unigenes were mapped into 118 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Differentially expressed genes (DEG) analysis identified 3, 16 and 5,899 DEGs from the comparison of 7Y vs. 15Y, 7Y vs. 21Y and 15Y vs. 21Y, respectively, in the immature xylem tissues, including 2,638 significantly up-regulated and 3,280 significantly down-regulated genes. Besides, five NAC transcription factors, 190 MYB transcription factors, and 34 WRKY transcription factors were identified respectively from Chinese fir transcriptome. Conclusion. Our results revealed the active transcriptional pathways and identified the DEGs at different cultivation phases of Chinese fir wood formation. This transcriptome dataset will aid in understanding and carrying out future studies on the molecular basis of Chinese fir wood formation and contribute to future artificial production and applications. PMID:27330860

Background Recent studies indicate that infestations of psocids pose a new risk for global food security. Among the psocids species, Liposcelis bostrychophila Badonnel has gained recognition in importance because of its parthenogenic reproduction, rapid adaptation, and increased worldwide distribution. To date, the molecular data available for L. bostrychophila is largely limited to genes identified through homology. Also, no transcriptome data relevant to psocids infection is available. Methodology and Principal Findings In this study, we generated de novo assembly of L. bostrychophila transcriptome performed through the short read sequencing technology (Illumina). In a single run, we obtained more than 51 million sequencing reads that were assembled into 60,012 unigenes (mean size = 711 bp) by Trinity. The transcriptome sequences from different developmental stages of L. bostrychophila including egg, nymph and adult were annotated with non-redundant (Nr) protein database, gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). The analysis revealed three major enzyme families involved in insecticide metabolism as differentially expressed in the L. bostrychophila transcriptome. A total of 49 P450-, 31 GST- and 21 CES-specific genes representing the three enzyme families were identified. Besides, 16 transcripts were identified to contain target site sequences of resistance genes. Furthermore, we profiled gene expression patterns upon insecticide (malathion and deltamethrin) exposure using the tag-based digital gene expression (DGE) method. Conclusion The L. bostrychophila transcriptome and DGE data provide gene expression data that would further our understanding of molecular mechanisms in psocids. In particular, the findings of this investigation will facilitate identification of genes involved in insecticide resistance and designing of new compounds for control of psocids. PMID:24278202

The most recent phylogenomic study suggested that Bryozoa (Ectoprocta), Brachiopoda, and Phoronida are monophyletic, implying that the lophophore of bryozoans, phoronids and brachiopods is a synapomorphy. Understanding the molecular mechanisms of the lophophore development of the Lophophorata clade can therefore provide us a new insight into the formation of the diverse morphological traits in metazoans. In the present study, we profiled the transcriptome of the Bryozoan (Ectoproct) Bugula neritina during the swimming larval stage (SW) and the early (4 h) and late (24 h) metamorphic stages using the Illumina HiSeq2000 platform. Various genes that function in development, the immune response and neurogenesis showed differential expression levels during metamorphosis. In situ hybridization of 23 genes that participate in the Wnt, BMP, Notch, and Hedgehog signaling pathways revealed their regulatory roles in the development of the lophophore and the ancestrula digestive tract. Our findings support the hypothesis that developmental precursors of the lophophore and the ancestrula digestive tract are pre-patterned by the differential expression of key developmental genes according to their fate. This study provides a foundation to better understand the developmental divergence and/or convergence among developmental precursors of the lophophore of bryozoans, branchiopods and phoronids. PMID:25300304

The most recent phylogenomic study suggested that Bryozoa (Ectoprocta), Brachiopoda, and Phoronida are monophyletic, implying that the lophophore of bryozoans, phoronids and brachiopods is a synapomorphy. Understanding the molecular mechanisms of the lophophore development of the Lophophorata clade can therefore provide us a new insight into the formation of the diverse morphological traits in metazoans. In the present study, we profiled the transcriptome of the Bryozoan (Ectoproct) Bugula neritina during the swimming larval stage (SW) and the early (4 h) and late (24 h) metamorphic stages using the Illumina HiSeq2000 platform. Various genes that function in development, the immune response and neurogenesis showed differential expression levels during metamorphosis. In situ hybridization of 23 genes that participate in the Wnt, BMP, Notch, and Hedgehog signaling pathways revealed their regulatory roles in the development of the lophophore and the ancestrula digestive tract. Our findings support the hypothesis that developmental precursors of the lophophore and the ancestrula digestive tract are pre-patterned by the differential expression of key developmental genes according to their fate. This study provides a foundation to better understand the developmental divergence and/or convergence among developmental precursors of the lophophore of bryozoans, branchiopods and phoronids. PMID:25300304

RNA localization is an important mechanism for achieving precise control of posttranscriptional gene expression. Previously, we demonstrated that a subset of cellular mRNAs copurify with mitotic microtubules in egg extracts of Xenopus laevis. Due to limited genomic sequence information available for X. laevis, we used RNA-seq to comprehensively identify the microtubule-interacting transcriptome of the related frog Xenopus tropicalis. We identified ∼450 mRNAs that showed significant enrichment on microtubules (MT-RNAs). In addition, we demonstrated that the MT-RNAs incenp, xrhamm, and tpx2 associate with spindle microtubules in vivo. MT-RNAs are enriched with transcripts associated with cell division, spindle formation, and chromosome function, demonstrating an overrepresentation of genes involved in mitotic regulation. To test whether uncharacterized MT-RNAs have a functional role in mitosis, we performed RNA interference and discovered that several MT-RNAs are required for normal spindle pole organization and γ-tubulin distribution. Together, these data demonstrate that microtubule association is one mechanism for compartmentalizing functionally related mRNAs within the nucleocytoplasmic space of mitotic cells and suggest that MT-RNAs are likely to contribute to spindle-localized mitotic translation. PMID:21937723

Background More than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE). Results We have sequenced 691,390 tags from four L-SAGE libraries increasing the existing gene expression data on cervical tissue by 20 fold. One-hundred and eighteen unique tags were highly expressed in normal cervical tissue and 107 of them mapped to unique genes, most belong to the ribosomal, calcium-binding and keratinizing gene families. We assessed these genes for aberrant expression in CIN III and five genes showed altered expression. In addition, we have identified twelve unique HPV 16 SAGE tags in the CIN III libraries absent in the normal libraries. Conclusion Establishing a baseline of gene expression in normal cervical tissue is key for identifying changes in cancer. We demonstrate the utility of this baseline data by identifying genes with aberrant expression in CIN III when compared to normal tissue. PMID:17543121

One of the major cardiovascular risk factor which predisposes to and accelerates atherosclerosis is arterial hypertension (AH). To determine the molecular basis of the crosslink between AH and atherosclerosis for the development of new treatment strategies large-scaletranscriptomeanalysis of the cells implicated in atherogenesis is needed. We used cDNA microarray technique for simultaneous analysis of gene expression in human abdominal aorta normal sites and atherosclerotic lesions of different histological types, as well as in peripheral blood leukocytes from patients with essential hypertension (EH) and donors. The microarray data were verified by quantitative RT-PCR (reverse transcription coupled with polymerase chain reaction) and immunohistochemical analysis. Differential expression of 40 genes has been found, among which twenty two genes demonstrated up-regulation and 18 genes demonstrated down-regulation in atherosclerotic aorta compared with normal vessel. New gene-candidates, implicated in atherogenesis, have been identified - FPRL2, CD37, CD53, RGS1, LCP1, SPI1, CTSA, EPAS1, FHL1, GEM, RHOB, SPARCL1, ITGA8, PLN, and COL14A1. These genes participate in cell migration and adhesion, phenotypic changes of smooth muscle cells, immune and inflammatory reactions, oxidative processes and extracellular matrix remodeling. We have found increased expression levels of CD53, SPI1, FPRL2, SPP1, CTSD, ACP5, LCP1, CTSA and LIPA genes in peripheral blood leukocytes from EH patients and in atherosclerotic lesions of human aorta. The majority of these genes significantly (p<0.005) positively (r>0.5) correlated with AH stage as well as with histological grading of atherosclerotic lesions. PMID:19772500

Background Variegation in flower color is commonly observed in many plant species and also occurs on ornamental peaches (Prunus persica f. versicolor [Sieb.] Voss). Variegated plants are highly valuable in the floricultural market. To gain a global perspective on genes differentially expressed in variegated peach flowers, we performed large-scaletranscriptome sequencing of white and red petals separately collected from a variegated peach tree. Results A total of 1,556,597 high-quality reads were obtained, with an average read length of 445 bp. The ESTs were assembled into 16,530 contigs and 42,050 singletons. The resulting unigenes covered about 60% of total predicted genes in the peach genome. These unigenes were further subjected to functional annotation and biochemical pathway analysis. Digital expression analysis identified a total of 514 genes differentially expressed between red and white flower petals. Since peach flower coloration is determined by the expression and regulation of structural genes relevant to flavonoid biosynthesis, a detailed examination detected four key structural genes, including C4H, CHS, CHI and F3H, expressed at a significantly higher level in red than in white petal. Except for the structural genes, we also detected 11 differentially expressed regulatory genes relating to flavonoid biosynthesis. Using the differentially expressed structural genes as the test objects, we validated the digital expression results by using quantitative real-time PCR, and the differential expression of C4H, CHS and F3H were confirmed. Conclusion In this study, we generated a large EST collection from flower petals of a variegated peach. By digital expression analysis, we identified an informative list of candidate genes associated with variegation in peach flowers, which offered a unique opportunity to uncover the genetic mechanisms underlying flower color variegation. PMID:24603808

A major impediment to the effective treatment of patients with pancreatic ductal adenocarcinoma (PDAC) is the molecular heterogeneity of this disease, which is reflected in an equally diverse pattern of clinical outcome and in responses to therapies. We developed an efficient strategy in which PDAC samples from 17 consecutive patients were collected by endoscopic ultrasound-guided fine-needle aspiration or surgery and were preserved as breathing tumors by xenografting and as a primary culture of epithelial cells. Transcriptomicanalysis was performed from breathing tumors by an Affymetrix approach. We observed significant heterogeneity in the RNA expression profile of tumors. However, the bioinformatic analysis of these data was able to discriminate between patients with long- and short-term survival corresponding to patients with moderately or poorly differentiated PDAC tumors, respectively. Primary culture of cells allowed us to analyze their relative sensitivity to anticancer drugs in vitro using a chemogram, similar to the antibiogram for microorganisms, establishing an individual profile of drug sensitivity. As expected, the response was patient dependent. We also found that transcriptomicanalysis predicts the sensitivity of cells to the five anticancer drugs most frequently used to treat patients with PDAC. In conclusion, using this approach, we found that transcriptomicanalysis could predict the sensitivity to anticancer drugs and the clinical outcome of patients with PDAC. PMID:25765988

A comprehensive transcriptomeanalysis of an expressed sequence tag (EST) database of the spider Dolomedes fimbriatus venom glands using single-residue distribution analysis (SRDA) identified 7,169 unique sequences. Mature chains of 163 different toxin-like polypeptides were predicted on the basis of well-established methodology. The number of protein precursors of these polypeptides was appreciably numerous than the number of mature polypeptides. A total of 451 different polypeptide precursors, translated from 795 unique nucleotide sequences, were deduced. A homology search divided the 163 mature polypeptide sequences into 16 superfamilies and 19 singletons. The number of mature toxins in a superfamily ranged from 2 to 49, whereas the diversity of the original nucleotide sequences was greater (2–261 variants). We observed a predominance of inhibitor cysteine knot toxin-like polypeptides among the cysteine-containing structures in the analyzed transcriptome bank. Uncommon spatial folds were also found. PMID:25977780

Monocytes and macrophages differentiate from progenitor cells under the influence of colony-stimulating factors. Genome-scale data have enabled the identification of the sets of genes that are associated with specific functions and the mechanisms by which thousands of genes are regulated in response to pathogen challenge. In large datasets, it is possible to identify large sets of genes that are coregulated with the transcription factors that regulate them. They include macrophage-specific genes, interferon-responsive genes, early inflammatory genes, and those associated with endocytosis. Such analyses can also extract macrophage-associated signatures from large cancer tissue datasets. However, cluster analysis provides no support for a signature that distinguishes macrophages from antigen-presenting dendritic cells, nor the classification of macrophage activation states as classical versus alternative, or M1 versus M2. Although there has been a focus on a small subset of lineage-enriched transcription factors, such as PU.1, more than half of the transcription factors in the genome can be expressed in macrophage lineage cells under some state of activation, and they interact in a complex network. The network architecture is conserved across species, but many of the target genes evolve rapidly and differ between mouse and human. The data and publication deluge related to macrophage biology require the development of new analytical tools and ways of presenting information in an accessible form. PMID:25319328

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes

Background Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. Methodology/Principal Findings We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Conclusions/Significance Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further

Background The entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterium, Photorhabdus luminescens, are important biological control agents of insect pests. This nematode-bacterium-insect association represents an emerging tripartite model for research on mutualistic and parasitic symbioses. Elucidation of mechanisms underlying these biological processes may serve as a foundation for improving the biological control potential of the nematode-bacterium complex. This large-scale expressed sequence tag (EST) analysis effort enables gene discovery and development of microsatellite markers. These ESTs will also aid in the annotation of the upcoming complete genome sequence of H. bacteriophora. Results A total of 31,485 high quality ESTs were generated from cDNA libraries of the adult H. bacteriophora TTO1 strain. Cluster analysis revealed the presence of 3,051 contigs and 7,835 singletons, representing 10,886 distinct EST sequences. About 72% of the distinct EST sequences had significant matches (E value < 1e-5) to proteins in GenBank's non-redundant (nr) and Wormpep190 databases. We have identified 12 ESTs corresponding to 8 genes potentially involved in RNA interference, 22 ESTs corresponding to 14 genes potentially involved in dauer-related processes, and 51 ESTs corresponding to 27 genes potentially involved in defense and stress responses. Comparison to ESTs and proteins of free-living nematodes led to the identification of 554 parasitic nematode-specific ESTs in H. bacteriophora, among which are those encoding F-box-like/WD-repeat protein theromacin, Bax inhibitor-1-like protein, and PAZ domain containing protein. Gene Ontology terms were assigned to 6,685 of the 10,886 ESTs. A total of 168 microsatellite loci were identified with primers designable for 141 loci. Conclusion A total of 10,886 distinct EST sequences were identified from adult H. bacteriophora cDNA libraries. BLAST searches revealed ESTs potentially involved in parasitism

Scorpions belonging to the Buthidae family have traditionally drawn much of the biochemist’s attention due to the strong toxicity of their venoms. Scorpions not toxic to mammals, however, also have complex venoms. They have been shown to be an important source of bioactive peptides, some of them identified as potential drug candidates for the treatment of several emerging diseases and conditions. It is therefore important to characterize the large diversity of components found in the non-Buthidae venoms. As a contribution to this goal, this manuscript reports the construction and characterization of cDNA libraries from four scorpion species belonging to the Vaejovis genus of the Vaejovidae family: Vaejovis mexicanus, V. intrepidus, V. subcristatus and V. punctatus. Some sequences coding for channel-acting toxins were found, as expected, but the main transcribed genes in the glands actively producing venom were those coding for non disulfide-bridged peptides. The ESTs coding for putative channel-acting toxins, corresponded to sodium channel β toxins, to members of the potassium channel-acting α or κ families, and to calcium channel-acting toxins of the calcin family. Transcripts for scorpine-like peptides of two different lengths were found, with some of the species coding for the two kinds. One sequence coding for La1-like peptides, of yet unknown function, was found for each species. Finally, the most abundant transcripts corresponded to peptides belonging to the long chain multifunctional NDBP-2 family and to the short antimicrobials of the NDBP-4 family. This apparent venom composition is in correspondence with the data obtained to date for other non-Buthidae species. Our study constitutes the first approach to the characterization of the venom gland transcriptome for scorpion species belonging to the Vaejovidae family. PMID:25659089

Lichen is a slow-growing symbiotic organism that consists of a fungus and a photobiont, comprising either an algae or a cyanobacterium living together in a single composite body, known as a thallus. Lichens have a remarkable ability to survive in extreme environmental conditions on earth that makes them a great biological indicator of air quality. The primary goal of this study is to discover the genes that may unravel the mechanism behind the tolerance of this lichen towards air pollution. Lichen samples of Dirinaria sp. were collected from two sites - Jerantut (J) as having a relatively good air quality and Klang (K), an area of bad air quality. Total RNA extraction was carried out, followed by sample preparation prior to transcriptomic sequencing. Altogether 21.7 million and 30.5 million high quality sequence reads from samples J and K, respectively were de novo assembled into 106884 and 88116 transcripts. The assembled sequences were annotated by BLASTX comparison against a non-redundant protein sequence database with 59403 sequences (67.4%) of sample K and 68972 sequences (64.5%) of sample J had a match in the database with a cut-off value of 1e-06. A total of 42175 sequences (47.8%) of sample K and 25648 sequences (24%) of sample J had a Gene Ontology term match. The sequences were assigned to Kyoto Encyclopedia of Genes and Genome (KEGG) pathways, resulting in 129 KEGG pathways generated from sample K, whilst 123 KEGG pathways were produced from sample J.

Scorpions belonging to the Buthidae family have traditionally drawn much of the biochemist's attention due to the strong toxicity of their venoms. Scorpions not toxic to mammals, however, also have complex venoms. They have been shown to be an important source of bioactive peptides, some of them identified as potential drug candidates for the treatment of several emerging diseases and conditions. It is therefore important to characterize the large diversity of components found in the non-Buthidae venoms. As a contribution to this goal, this manuscript reports the construction and characterization of cDNA libraries from four scorpion species belonging to the Vaejovis genus of the Vaejovidae family: Vaejovis mexicanus, V. intrepidus, V. subcristatus and V. punctatus. Some sequences coding for channel-acting toxins were found, as expected, but the main transcribed genes in the glands actively producing venom were those coding for non disulfide-bridged peptides. The ESTs coding for putative channel-acting toxins, corresponded to sodium channel β toxins, to members of the potassium channel-acting α or κ families, and to calcium channel-acting toxins of the calcin family. Transcripts for scorpine-like peptides of two different lengths were found, with some of the species coding for the two kinds. One sequence coding for La1-like peptides, of yet unknown function, was found for each species. Finally, the most abundant transcripts corresponded to peptides belonging to the long chain multifunctional NDBP-2 family and to the short antimicrobials of the NDBP-4 family. This apparent venom composition is in correspondence with the data obtained to date for other non-Buthidae species. Our study constitutes the first approach to the characterization of the venom gland transcriptome for scorpion species belonging to the Vaejovidae family. PMID:25659089

The Sedum alfredii Hance hyperaccumulating ecotype (HE) has the ability to hyperaccumulate cadmium (Cd), as well as zinc (Zn) and lead (Pb) in above-ground tissues. Although many physiological studies have been conducted with these plants, the molecular mechanisms underlying their hyper-tolerance to heavy metals are largely unknown. Here we report on the generation of 9.4 gigabases of adaptor-trimmed raw sequences and the assembly of 57,162 transcript contigs in S. alfredii Hance (HE) shoots by the combination of Roche 454 and Illumina/Solexa deep sequencing technologies. We also have functionally annotated the transcriptome and analyzed the transcriptome changes upon Cd hyperaccumulation in S. alfredii Hance (HE) shoots. There are 110 contigs and 123 contigs that were up-regulated (Fold Change ≧2.0) and down-regulated (Fold Change ≦0.5) by chronic Cd treatment in S. alfredii Hance (HE) at q-value cutoff of 0.005, respectively. Quantitative RT-PCR was employed to compare gene expression patterns between S. alfredii Hance (HE) and non-hyperaccumulating ecotype (NHE). Our results demonstrated that several genes involved in cell wall modification, metal translocation and remobilization were more induced or constitutively expressed at higher levels in HE shoots than that in NHE shoots in response to Cd exposure. Together, our study provides large-scale expressed sequence information and genome-wide transcriptome profiling of Cd responses in S. alfredii Hance (HE) shoots. PMID:23755133

The Sedum alfredii Hance hyperaccumulating ecotype (HE) has the ability to hyperaccumulate cadmium (Cd), as well as zinc (Zn) and lead (Pb) in above-ground tissues. Although many physiological studies have been conducted with these plants, the molecular mechanisms underlying their hyper-tolerance to heavy metals are largely unknown. Here we report on the generation of 9.4 gigabases of adaptor-trimmed raw sequences and the assembly of 57,162 transcript contigs in S. alfredii Hance (HE) shoots by the combination of Roche 454 and Illumina/Solexa deep sequencing technologies. We also have functionally annotated the transcriptome and analyzed the transcriptome changes upon Cd hyperaccumulation in S. alfredii Hance (HE) shoots. There are 110 contigs and 123 contigs that were up-regulated (Fold Change ≥ 2.0) and down-regulated (Fold Change =0.5) by chronic Cd treatment in S. alfredii Hance (HE) at q-value cutoff of 0.005, respectively. Quantitative RT-PCR was employed to compare gene expression patterns between S. alfredii Hance (HE) and non-hyperaccumulating ecotype (NHE). Our results demonstrated that several genes involved in cell wall modification, metal translocation and remobilization were more induced or constitutively expressed at higher levels in HE shoots than that in NHE shoots in response to Cd exposure. Together, our study provides large-scale expressed sequence information and genome-wide transcriptome profiling of Cd responses in S. alfredii Hance (HE) shoots. PMID:23755133

The extensive data on the transcription of the plant genome are derived primarily from the sporophytic generation. There currently is little information on genes that are expressed during female gametophyte development in angiosperms, and it is not known whether the female gametophyte transcriptome contains a major set of genes that are not expressed in the sporophyte or whether it is primarily a subset of the sporophytic transcriptome. Because the embryo sac is embedded within the maternal ovule tissue, we have utilized the Arabidopsis (Arabidopsis thaliana) mutant sporocyteless that produces ovules without embryo sacs, together with the ATH1 Arabidopsis whole-genome oligonucleotide array, to identify genes that are preferentially or specifically expressed in female gametophyte development. From analysis of the datasets, 225 genes are identified as female gametophyte genes, likely a lower limit as stringent criteria were used for the analysis, eliminating many low expressed genes. Nearly 45% of the identified genes were not previously detected by sporophytic expression profiling, suggesting that the embryo sac transcriptome may contain a significant fraction of transcripts restricted to the gametophyte. Validation of six candidate genes was performed using promoter∷β-glucuronidase fusions, and all of these showed embryo sac-specific expression in the ovule. The unfiltered expression data from this study can be used to evaluate the possibility of female gametophytic expression for any gene in the ATH1 array, and contribute to identification of the functions of the component of the Arabidopsis genome not represented in studies of sporophytic expression and function. PMID:16299181

Porphyromonas gingivalis is a major etiological agent in chronic and aggressive forms of periodontal disease. The organism is an asaccharolytic anaerobe and is a constituent of mixed species biofilms in a variety of microenvironments in the oral cavity. P. gingivalis expresses a range of virulence factors over which it exerts tight control. High-throughput sequencing technologies provide the opportunity to relate functional genomics to basic biology. In this study we report qualitative and quantitative RNA-Seq analysis of the transcriptome of P. gingivalis. We have also applied RNA-Seq to the transcriptome of a ΔluxS mutant of P. gingivalis deficient in AI-2-mediated bacterial communication. The transcriptomeanalysis confirmed the expression of all predicted ORFs for strain ATCC 33277, including 854 hypothetical proteins, and allowed the identification of hitherto unknown transcriptional units. Twelve non-coding RNAs were identified, including 11 small RNAs and one cobalamin riboswitch. Fifty-seven genes were differentially regulated in the LuxS mutant. Addition of exogenous synthetic 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor) to the ΔluxS mutant culture complemented expression of a subset of genes, indicating that LuxS is involved in both AI-2 signaling and non-signaling dependent systems in P. gingivalis. This work provides an important dataset for future study of P. gingivalis pathophysiology and further defines the LuxS regulon in this oral pathogen. PMID:22919670

Verticillium wilt is one of the main diseases in cotton (Gossypium hirsutum), severely reduces yield and fiber quality, and is difficult to be con-trolled effectively. At present, the molecular mechanism that confers resistance to this disease is unclear. Transcriptome sequencing is an important method to detect resistance genes, explore metabolic pathways, and study resistance mechanisms. In this study, the transcriptome of a disease-resistant inbred cot-ton line inoculated with Verticillium dahliae was sequenced. A total of 126,402 unigenes were obtained using de novo assembly and data analysis, 99,712 (78.88%) of which were annotated into the Nr, Nt, Swiss-Prot, KEGG, COG, and GO databases. The expression patterns of 16 candidate disease-resis-tance genes showed that some genes were upregulated soon after V. dahliae inoculation and others were upregulated later, which may indicate instanta-neous basal defense and lagged specific defense, respectively. We conducted a preliminary analysis of the transcriptome database, which will contribute to further research regarding the cloning of disease-resistance genes. PMID:26535625

Identification of the genetic events that contribute to host-pathogen interactions is important for understanding the natural history of infectious diseases and developing therapeutics. Transcriptome studies conducted on pathogens have been central to this goal in recent years. However, most of these investigations have focused on specific end points or disease phases, rather than analysis of the entire time course of infection. To gain a more complete understanding of how bacterial gene expression changes over time in a primate host, the transcriptome of group A Streptococcus (GAS) was analyzed during an 86-day infection protocol in 20 cynomolgus macaques with experimental pharyngitis. The study used 260 custom Affymetrix (Santa Clara, CA) chips, and data were confirmed by TaqMan analysis. Colonization, acute, and asymptomatic phases of disease were identified. Successful colonization and severe inflammation were significantly correlated with an early onset of superantigen gene expression. The differential expression of two-component regulators covR and spy0680 (M1_spy0874) was significantly associated with GAS colony-forming units, inflammation, and phases of disease. Prophage virulence gene expression and prophage induction occurred predominantly during high pathogen cell densities and acute inflammation. We discovered that temporal changes in the GAS transcriptome were integrally linked to the phase of clinical disease and host-defense response. Knowledge of the gene expression patterns characterizing each phase of pathogen-host interaction provides avenues for targeted investigation of proven and putative virulence factors and genes of unknown function and will assist vaccine research. PMID:15956184

Molecular mechanisms mediating group A Streptococcus (GAS)-host interactions remain poorly understood but are crucial for diagnostic, therapeutic, and vaccine development. An optimized high-density microarray was used to analyze the transcriptome of GAS during experimental mouse soft tissue infection. The transcriptome of a wild-type serotype M1 GAS strain and an isogenic transcriptional regulator knockout mutant (covR) also were compared. Array datasets were verified by quantitative real-time reverse transcriptase-polymerase chain reaction and in situ immunohistochemistry. The results unambiguously demonstrate that coordinated expression of proven and putative GAS virulence factors is directed toward overwhelming innate host defenses leading to severe cellular damage. We also identified adaptive metabolic responses triggered by nutrient signals and hypoxic/acidic conditions in the host, likely facilitating pathogen persistence and proliferation in soft tissues. Key discoveries included that oxidative stress genes, virulence genes, genes related to amino acid and maltodextrin utilization, and several two-component transcriptional regulators were highly expressed in vivo. This study is the first global analysis of the GAS transcriptome during invasive infection. Coupled with parallel analysis of the covR mutant strain, novel insights have been made into the regulation of GAS virulence in vivo, resulting in new avenues for targeted therapeutic and vaccine research. PMID:16936267

We developed phase-switch microfluidic devices for molecular profiling of a large number of single cells. Whole genome microarrays and RNA-sequencing are commonly used to determine the expression levels of genes in cell lysates (a physical mix of millions of cells) for inferring gene functions. However, cellular heterogeneity becomes an inherent noise in the measurement of gene expression. The unique molecular characteristics of individual cells, as well as the temporal and quantitative information of gene expression in cells, are lost when averaged among all cells in cell lysates. Our single-cell technology overcomes this limitation and enables us to obtain a large number of single-cell transcriptomes from a population of cells. A collection of single-cell molecular profiles allows us to study carcinogenesis from an evolutionary perspective by treating cancer as a diverse population of cells with abnormal molecular characteristics. Because a cancer cell population contains cells at various stages of development toward drug resistance, clustering similar single-cell molecular profiles could reveal how drug-resistant sub-clones evolve during cancer treatment. Here, we discuss how single-cell transcriptomeanalysis technology could enable the study of carcinogenesis from an evolutionary perspective and the development of drug-resistance in leukemia. The single-cell transcriptomeanalysis reported here could have a direct and significant impact on current cancer treatments and future personalized cancer therapies. PMID:23706768

Sex-biased genes are considered to account for most of phenotypic differences between males and females. In order to explore the sex-biased gene expression in crab, we performed the whole-body transcriptomeanalysis in male and female juveniles of the Chinese mitten crab Eriocheir sinensis using next-generation sequencing technology. Of the 23,349 annotated unigenes, 148 were identified as sex-related genes. A total of 29 candidate genes involved in primary sex determination pathways were detected, indicating the sex determination cascade of the mitten crab might be more complex than previously supposed. Differential expression analysis showed 448 differentially expressed genes (DEGs) between the two transcriptomes. Most of DEGs were involved in processes such as metabolism and immunity, and not associated with obvious sexual function. The pathway predominantly enriched for DEGs were related to lysosome, which might reflect the differences in metabolism between males and females. Of the immune DGEs, 18 up-regulated genes in females were humoral immune factors, and eight up-regulated genes in males were pattern recognition receptors, suggesting sex differences of immune defense might exist in the mitten crab. In addition, two reproduction-related genes, vitellogenin and insulin-like androgenic gland factor, were identified to express in both sexes but with significantly higher level in males. Our research provides the first whole-body RNA sequencing of sex-specific transcriptomes for juvenile E. sinensis and will facilitate further studies on molecular mechanisms of crab sexual dimorphism. PMID:26193085

Constraint-based metabolic modeling such as flux balance analysis (FBA) has been widely used to simulate cell metabolism. Thanks to its simplicity and flexibility, numerous algorithms have been developed based on FBA and successfully predicted the phenotypes of various biological systems. However, their phenotype predictions may not always be accurate in FBA because of using the objective function that is assumed for cell metabolism. To overcome this challenge, we have developed a novel computational framework, namely omFBA, to integrate multi-omics data (e.g. transcriptomics) into FBA to obtain omics-guided objective functions with high accuracy. In general, we first collected transcriptomics data and phenotype data from published database (e.g. GEO database) for different microorganisms such as Saccharomyces cerevisiae. We then developed a “Phenotype Match” algorithm to derive an objective function for FBA that could lead to the most accurate estimation of the known phenotype (e.g. ethanol yield). The derived objective function was next correlated with the transcriptomics data via regression analysis to generate the omics-guided objective function, which was next used to accurately simulate cell metabolism at unknown conditions. We have applied omFBA in studying sugar metabolism of S. cerevisiae and found that the ethanol yield could be accurately predicted in most of the cases tested (>80%) by using transcriptomics data alone, and revealed valuable metabolic insights such as the dynamics of flux ratios. Overall, omFBA presents a novel platform to potentially integrate multi-omics data simultaneously and could be incorporated with other FBA-derived tools by replacing the arbitrary objective function with the omics-guided objective functions. PMID:27100883

The Antarctic green alga Chlamydomonas sp. ICE-L was isolated from sea ice. As a psychrophilic microalga, it can tolerate the environmental stress in the sea-ice brine, such as freezing temperature and high salinity. We performed a transcriptomeanalysis to identify freezing stress responding genes and explore the extreme environmental acclimation-related strategies. Here, we show that many genes in ICE-L transcriptome that encoding PUFA synthesis enzymes, molecular chaperon proteins, and cell membrane transport proteins have high similarity to the gens from Antarctic bacteria. These ICE-L genes are supposed to be acquired through horizontal gene transfer from its symbiotic microbes in the sea-ice brine. The presence of these genes in both sea-ice microalgae and bacteria indicated the biological processes they involved in are possibly contributing to ICE-L success in sea ice. In addition, the biological pathways were compared between ICE-L and its closely related sister species, Chlamydomonas reinhardtii and Volvox carteri. In ICE-L transcripome, many sequences homologous to the plant or bacteria proteins in the post-transcriptional, post-translational modification, and signal-transduction KEGG pathways, are absent in the nonpsychrophilic green algae. These complex structural components might imply enhanced stress adaptation capacity. At last, differential gene expression analysis at the transcriptome level of ICE-L indicated that genes that associated with post-translational modification, lipid metabolism, and nitrogen metabolism are responding to the freezing treatment. In conclusion, the transcriptome of Chlamydomonas sp. ICE-L is very useful for exploring the mutualistic interaction between microalgae and bacteria in sea ice; and discovering the specific genes and metabolism pathways responding to the freezing acclimation in psychrophilic microalgae. PMID:27161450

Skeletal muscle fibers are mainly categorized into red and white fiber types, and the ratio of red/white fibers within muscle mass plays a crucial role in meat quality such as tenderness and flavor. To better understand the molecular difference between the two muscle fibers, this study takes advantage of RNA-seq to compare differences in the transcriptome between extensor digitorum longus (EDL; white fiber) and soleus (Sol; red fiber) muscles of large white pigs. In total, 89,658,562 and 46,723,568 raw reads from EDL and Sol were generated, respectively. Comparison between the two transcriptomes revealed 561 differentially expressed genes, with 408 displaying higher and 153 lower levels of expression in Sol. Quantitative real-time polymerase chain reaction validated the differential expression of nine genes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis discovered several differentially enriched biological functions and processes of the two muscles. Moreover, transcriptome comparison between EDL and Sol identified many muscle-related genes (CSRP3, ACTN2, MYL1, and MYH6) and pathways related to myofiber formation, such as focal adhesion, tight junction formation, extracellular matrix (ECM)-receptor pathway, calcium signaling, and Wnt signaling. In addition, 58,362 and 58,359 single nucleotide polymorphisms were identified in EDL and Sol, respectively, and the sequence of 9069 genes was refined at the 5', 3' or both ends. Numerous novel transcripts and alternatively spliced RNAs were also identified. Our transcriptomeanalysis constitutes valuable sequence resource for uncovering important genes and pathways involved in muscle fiber type determination, and might help further our understanding of the molecular mechanisms in different types of muscle. PMID:26520103

Tick salivary gland (SG) proteins possess powerful pharmacologic properties that facilitate tick feeding and pathogen transmission. For the first time, SG transcriptomes of Ixodes ricinus, an important disease vector for humans and animals, were analyzed using next-generation sequencing. SGs were collected from different tick life stages fed on various animal species, including cofeeding of nymphs and adults on the same host. Four cDNA samples were sequenced, discriminating tick SG transcriptomes of early- and late-feeding nymphs or adults. In total, 441,381,454 pyrosequencing reads and 67,703,183 Illumina reads were assembled into 272,220 contigs, of which 34,560 extensively annotated coding sequences are disclosed; 8686 coding sequences were submitted to GenBank. Overall, 13% of contigs were classified as secreted proteins that showed significant differences in the transcript representation among the 4 SG samples, including high numbers of sample-specific transcripts. Detailed phylogenetic reconstructions of two relatively abundant SG-secreted protein families demonstrated how this study improves our understanding of the molecular evolution of hematophagy in arthropods. Our data significantly increase the available genomic information for I. ricinus and form a solid basis for future tick genome/transcriptome assemblies and the functional analysis of effectors that mediate the feeding physiology and parasite-vector interaction of I. ricinus.—Schwarz, A., von Reumont, B.M., Erhart, J., Chagas, A.C., Ribeiro, J.M.C., Kotsyfakis, M. De novo Ixodes ricinus salivary gland transcriptomeanalysis using two next-generation sequencing methodologies. PMID:23964076

Background As an arborescent and perennial plant, Moso bamboo (Phyllostachys edulis (Carrière) J. Houzeau, synonym Phyllostachys heterocycla Carrière) is characterized by its infrequent sexual reproduction with flowering intervals ranging from several to more than a hundred years. However, little bamboo genomic research has been conducted on this due to a variety of reasons. Here, for the first time, we investigated the transcriptome of developing flowers in Moso bamboo by using high-throughput Illumina GAII sequencing and mapping short reads to the Moso bamboo genome and reference genes. We performed RNA-seq analysis on four important stages of flower development, and obtained extensive gene and transcript abundance data for the floral transcriptome of this key bamboo species. Results We constructed a cDNA library using equal amounts of RNA from Moso bamboo leaf samples from non-flowering plants (CK) and mixed flower samples (F) of four flower development stages. We generated more than 67 million reads from each of the CK and F samples. About 70% of the reads could be uniquely mapped to the Moso bamboo genome and the reference genes. Genes detected at each stage were categorized to putative functional categories based on their expression patterns. The analysis of RNA-seq data of bamboo flowering tissues at different developmental stages reveals key gene expression properties during the flower development of bamboo. Conclusion We showed that a combination of transcriptome sequencing and RNA-seq analysis was a powerful approach to identifying candidate genes related to floral transition and flower development in bamboo species. The results give a better insight into the mechanisms of Moso bamboo flowering and ageing. This transcriptomic data also provides an important gene resource for improving breeding for Moso bamboo. PMID:24915141

Confounding factors In transcriptomics experimentation, confounding factors frequently exist alongside the intended experimental factors and can severely influence the outcome of a transcriptomeanalysis. Confounding factors are regularly discussed in methodological literature, but their actual, practical impact on the outcome and interpretation of transcriptomics experiments is, to our knowledge, not documented. For instance, in-vivo experimental factors; like Individual, Sample-Composition and Time-of-Day are potentially formidable confounding factors. To study these confounding factors, we designed an extensive in-vivo transcriptome experiment (n = 264) with UVR exposure of murine skin containing six consecutive samples from each individual mouse (n = 64). Analysis Approach Evaluation of the confounding factors: Sample-Composition, Time-of-Day, Handling-Stress, and Individual-Mouse resulted in the identification of many genes that were affected by them. These genes sometimes showed over 30-fold expression differences. The most prominent confounding factor was Sample-Composition caused by mouse-dependent skin composition differences, sampling variation and/or influx/efflux of mobile cells. Although we can only evaluate these effects for known cell type specifically expressed genes in our complex heterogeneous samples, it is clear that the observed variations also affect the cumulative expression levels of many other non-cell-type-specific genes. ANOVA ANOVA analysis can only attempt to neutralize the effects of the well-defined confounding factors, such as Individual-Mouse, on the experimental factors UV-Dose and Recovery-Time. Also, by definition, ANOVA only yields reproducible gene-expression differences, but we found that these differences were very small compared to the fold changes induced by the confounding factors, questioning the biological relevance of these ANOVA-detected differences. Furthermore, it turned out that many of the differentially expressed

Specific gene expression in oocytes and its surrounding cumulus (CC) and granulosa (GC) cells is needed for successful folliculogenesis and oocyte maturation. The aim of the present study was to compare genome-wide gene expression and biological functions of human GC and CC. Individual GC and CC were derived from 37 women undergoing IVF procedures. Gene expression analysis was performed using microarrays, followed by a meta-analysis. Results were validated using quantitative real-time PCR. There were 6029 differentially expressed genes (q < 10−4); of which 650 genes had a log2 FC ≥ 2. After the meta-analysis there were 3156 genes differentially expressed. Among these there were genes that have previously not been reported in human somatic follicular cells, like prokineticin 2 (PROK2), higher expressed in GC, and pregnancy up-regulated nonubiquitous CaM kinase (PNCK), higher expressed in CC. Pathways like inflammatory response and angiogenesis were enriched in GC, whereas in CC, cell differentiation and multicellular organismal development were among enriched pathways. In conclusion, transcriptomes of GC and CC as well as biological functions, are distinctive for each cell subpopulation. By describing novel genes like PROK2 and PNCK, expressed in GC and CC, we upgraded the existing data on human follicular biology. PMID:26313571

Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37–100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins. PMID:26284934

Cockroaches are surrogate hosts for microbes that cause many human diseases. In spite of their generally destructive nature, cockroaches have recently been found to harbor potentially beneficial and medically useful substances such as drugs and allergens. However, genomic information for the American cockroach (Periplaneta americana) is currently unavailable; therefore, transcriptome and gene expression profiling is needed as an important resource to better understand the fundamental biological mechanisms of this species, which would be particularly useful for the selection of novel antimicrobial peptides. Thus, we performed de novo transcriptomeanalysis of P. americana that were or were not immunized with Escherichia coli. Using an Illumina HiSeq sequencer, we generated a total of 9.5 Gb of sequences, which were assembled into 85,984 contigs and functionally annotated using Basic Local Alignment Search Tool (BLAST), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) database terms. Finally, using an in silico antimicrobial peptide prediction method, 86 antimicrobial peptide candidates were predicted from the transcriptome, and 21 of these peptides were experimentally validated for their antimicrobial activity against yeast and gram positive and -negative bacteria by a radial diffusion assay. Notably, 11 peptides showed strong antimicrobial activities against these organisms and displayed little or no cytotoxic effects in the hemolysis and cell viability assay. This work provides prerequisite baseline data for the identification and development of novel antimicrobial peptides, which is expected to provide a better understanding of the phenomenon of innate immunity in similar species. PMID:27167617

Cockroaches are surrogate hosts for microbes that cause many human diseases. In spite of their generally destructive nature, cockroaches have recently been found to harbor potentially beneficial and medically useful substances such as drugs and allergens. However, genomic information for the American cockroach (Periplaneta americana) is currently unavailable; therefore, transcriptome and gene expression profiling is needed as an important resource to better understand the fundamental biological mechanisms of this species, which would be particularly useful for the selection of novel antimicrobial peptides. Thus, we performed de novo transcriptomeanalysis of P. americana that were or were not immunized with Escherichia coli. Using an Illumina HiSeq sequencer, we generated a total of 9.5 Gb of sequences, which were assembled into 85,984 contigs and functionally annotated using Basic Local Alignment Search Tool (BLAST), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) database terms. Finally, using an in silico antimicrobial peptide prediction method, 86 antimicrobial peptide candidates were predicted from the transcriptome, and 21 of these peptides were experimentally validated for their antimicrobial activity against yeast and gram positive and -negative bacteria by a radial diffusion assay. Notably, 11 peptides showed strong antimicrobial activities against these organisms and displayed little or no cytotoxic effects in the hemolysis and cell viability assay. This work provides prerequisite baseline data for the identification and development of novel antimicrobial peptides, which is expected to provide a better understanding of the phenomenon of innate immunity in similar species. PMID:27167617

Recent studies in domestic animals have used RNA-seq to explore the transcriptome of different tissues in a limited number of individuals. In the present study, de novo transcriptome sequencing was used to compare sheep adipose tissue transcriptome profiles between a fat-tailed breed (Kazak sheep; KS) and a short-tailed (Tibetan sheep; TS). The RNA-seq data from these two groups revealed that 646 genes were differentially expressed between the KS and TS groups, including 280 up-regulated and 366 down-regulated genes. We identified genes relevant to fat metabolism in adipose tissues, including two top genes with the largest fold change (NELL1 and FMO3). Pathway analysis revealed that the differentially expressed genes between the KS and TS breeds belong to fatty acid metabolism relevant pathways (e.g. fat digestion and absorption, glycine, serine, and threonine metabolism) and cell junction-related pathways (e.g. cell adhesion molecules) which contribute to fat deposition. This work highlighted potential genes and gene networks that affect fat deposition and meat quality in sheep. PMID:25088569

Raphanus sativus is not only a popular edible vegetable but also an important source of medicinal compounds. However, the paucity of knowledge about the transcriptome of R. sativus greatly impedes better understanding of the functional genomics and medicinal potential of R. sativus. In this study, the transcriptome sequencing of leaf tissues in R. sativus was performed for the first time. Approximately 22 million clean reads were generated and used for transcriptome assembly. The generated unigenes were subsequently annotated against gene ontology (GO) database. KEGG analysis further revealed two important pathways in the bolting stage of R.sativus including spliceosome assembly and alkaloid synthesis. In addition, a total of 6,295 simple sequence repeats (SSRs) with various motifs were identified in the unigene library of R. sativus. Finally, four unigenes of R. sativus were selected for alignment with their homologs from other plants, and phylogenetic trees for each of the genes were constructed. Taken together, this study will provide a platform to facilitate gene discovery and advance functional genomic research of R. sativus. PMID:24265813

Fruit formation and early development involve a range of physiological and morphological transformations of the various constituent tissues of the ovary. These developmental changes vary considerably according to tissue type, but molecular analyses at an organ-wide level inevitably obscure many tissue-specific phenomena. We used laser-capture microdissection coupled to high-throughput RNA sequencing to analyze the transcriptome of ovaries and fruit tissues of the wild tomato species Solanum pimpinellifolium. This laser-capture microdissection-high-throughput RNA sequencing approach allowed quantitative global profiling of gene expression at previously unobtainable levels of spatial resolution, revealing numerous contrasting transcriptome profiles and uncovering rare and cell type-specific transcripts. Coexpressed gene clusters linked specific tissues and stages to major transcriptional changes underlying the ovary-to-fruit transition and provided evidence of regulatory modules related to cell division, photosynthesis, and auxin transport in internal fruit tissues, together with parallel specialization of the pericarp transcriptome in stress responses and secondary metabolism. Analysis of transcription factor expression and regulatory motifs indicated putative gene regulatory modules that may regulate the development of different tissues and hormonal processes. Major alterations in the expression of hormone metabolic and signaling components illustrate the complex hormonal control underpinning fruit formation, with intricate spatiotemporal variations suggesting separate regulatory programs. PMID:26099271

Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins. PMID:26284934

Wheat (Triticum aestivum L.) cultivars possessing purple grain arethought to be more nutritious because of high anthocyanin contents in the pericarp. Comparative transcriptomeanalysis of purple (cv Gy115) and white pericarps was carried out using next-generation sequencing technology. There were 23,642 unigenes significantly differentially expressed in the purple and white pericarps, including 9945 up-regulated and 13,697 down-regulated. The differentially expressed unigenes were mainly involved in encoding components of metabolic pathways, The flavonoid biosynthesis pathway was the most represented in metabolic pathways. In the transcriptome of purple pericarp in Gy115, most structural and regulatory genes biosynthesizing anthocyanin were identified, and had higher expression levels than in white pericarp. The largestunigene of anthocyanin biosynthesis in Gy115 was longer than the reference genes, which implies that high-throughput sequencing could isolate the genes of anthocyanin biosynthesis in tissues or organs with high anthocyanin content. Based on present and previous results, three unigenes of MYB gene on chromosome 7BL and three unigenes of MYC on chromosome 2AL were predicted as candidate genes for the purple grain trait. This article was the first to provide a systematic overview comparing the transcriptomes of purple and white pericarps in common wheat, which should be very valuable for identifying the key genes for the purple pericarp trait. PMID:27171148

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by the deficiency of β-glucuronidase. In this study, we compared the changes relative to normal littermates in the proteome and transcriptome of the hippocampus in the C57Bl/6 mouse model of MPS VII, which has well-documented histopathological and neurodegenerative changes. A completely different set of significant changes between normal and MPS VII littermates were found in each assay. Nevertheless, the functional annotation terms generated by the two methods showed agreement in many of the processes, which also corresponded to known pathology associated with the disease. Additionally, assay-specific changes were found, which in the proteomic analysis included mitochondria, energy generation, and cytoskeletal differences in the mutant, while the transcriptome differences included immune, vesicular, and extracellular matrix changes. In addition, the transcriptomic changes in the mutant hippocampus were concordant with those in a MPS VII mouse caused by the same mutation but on a different background inbred strain. PMID:27053151

The planarian Schmidtea mediterranea is a powerful model organism for studying stem cell biology due to its extraordinary regenerative ability mediated by neoblasts, a population of adult somatic stem cells. Elucidation of the S. mediterranea transcriptome and the dynamics of transcript expression will increase our understanding of the gene regulatory programs that regulate stem cell function and differentiation. Here, we have used RNA-Seq to characterize the S. mediterranea transcriptome in sexual and asexual animals and in purified neoblast and differentiated cell populations. Our analysis identified many uncharacterized genes, transcripts, and alternatively spliced isoforms that are differentially expressed in a strain or cell type-specific manner. Transcriptome profiling of purified neoblasts and differentiated cells identified neoblast-enriched transcripts, many of which likely play important roles in regeneration and stem cell function. Strikingly, many of the neoblast-enriched genes are orthologs of genes whose expression is enriched in human embryonic stem cells, suggesting that a core set of genes that regulate stem cell function are conserved across metazoan species. PMID:22496805

Background Whitefly (Bemisia tabaci) causes extensive crop damage throughout the world by feeding directly on plants and by vectoring hundreds of species of begomoviruses. Yet little is understood about its genes involved in development, insecticide resistance, host range plasticity and virus transmission. Results To facilitate research on whitefly, we present a method for de novo assembly of whitefly transcriptome using short read sequencing technology (Illumina). In a single run, we produced more than 43 million sequencing reads. These reads were assembled into 168,900 unique sequences (mean size = 266 bp) which represent more than 10-fold of all the whitefly sequences deposited in the GenBank (as of March 2010). Based on similarity search with known proteins, these analyses identified 27,290 sequences with a cut-off E-value above 10-5. Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. In addition, we investigated the transcriptome changes during whitefly development using a tag-based digital gene expression (DGE) system. We obtained a sequencing depth of over 2.5 million tags per sample and identified a large number of genes associated with specific developmental stages and insecticide resistance. Conclusion Our data provides the most comprehensive sequence resource available for whitefly study and demonstrates that the Illumina sequencing allows de novo transcriptome assembly and gene expression analysis in a species lacking genome information. We anticipate that next generation sequencing technologies hold great potential for the study of the transcriptome in other non-model organisms. PMID:20573269

Background The lack of sequenced genomes for oleaginous microalgae limits our understanding of the mechanisms these organisms utilize to become enriched in triglycerides. Here we report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous microalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG) biofuel precursor. Results After growth under nitrogen replete and nitrogen limiting conditions, we quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant pathways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite measurements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during nitrogen limitation was bolstered by repression of the β-oxidation pathway, up-regulation of genes encoding for the pyruvate dehydrogenase complex which funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid biosynthesis, and the up-regulation of lipases—presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis. Conclusions Our quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and provides a variety of genetic

Background Preeclampsia (PE) is a serious hypertensive pregnancy disorder with a significant genetic component. Numerous genetic studies, including our own, have yielded many susceptibility genes from distinct functional groups. Additionally, transcriptome profiling of tissues at the maternal-fetal interface has likewise yielded many differentially expressed genes. Often there is little overlap between these two approaches, although genes identified in both approaches are significantly associated with PE. We have thus taken a novel integrative bioinformatics approach of analysing pathways common to the susceptibility genes and the PE transcriptome. Methods Using Illumina Human Ht12v4 and Wg6v3 BeadChips, transcriptome profiling was conducted on n = 65 normotensive and n = 60 PE decidua basalis tissues collected at delivery. The R software package libraries lumi and limma were used to preprocess transcript data for pathway analysis. Pathways were analysed and constructed using Pathway Studio. We examined ten candidate genes, which are from these functional groups: activin/inhibin signalling—ACVR1, ACVR1C, ACVR2A, INHA, INHBB; structural components—COL4A1, COL4A2 and M1 family aminopeptidases—ERAP1, ERAP2 and LNPEP. Results/Conclusion Major common regulators/targets of these susceptibility genes identified were AGT, IFNG, IL6, INHBA, SERPINE1, TGFB1 and VEGFA. The top two categories of pathways associated with the susceptibility genes, which were significantly altered in the PE decidual transcriptome, were apoptosis and cell signaling (p < 0.001). Thus, susceptibility genes from distinct functional groups share similar downstream pathways through common regulators/targets, some of which are altered in PE. This study contributes to a better understanding of how susceptibility genes may interact in the development of PE. With this knowledge, more targeted functional analyses of PE susceptibility genes in these key pathways can be performed to examine their

Rumen flukes are parasitic trematodes (Platyhelminthes: Digenea) of major socioeconomic importance in many countries. Key representatives, such as Paramphistomum cervi, can cause "Rumen fluke disease" or paramphistomosis and undermine economic animal productivity and welfare. P. cervi is primarily a problem in sheep, goat and buffalo production as a consequence of reduced weight gain and milk production, clinical disease or death. Recent technological advances in genomics and bioinformatics now provide unique opportunities for the identification and pre-validation of drug targets and vaccines through improved understanding of the biology of pathogens such as P. cervi and their relationship with their hosts at the molecular level. Here, we report next generation transcriptome sequencing analysis for P. cervi. RNAseq libraries were generated from RNA extracted from 15 adult P. cervi parasites sampled from each of three different host species (sheep, goat and buffalo) and a reference transcriptome was generated by assembly of all Ion Torrent PGM sequencing data. Raw reads (7,433,721 in total) were initially filtered for host nucleotide contamination and ribosomal RNAs and the remaining reads were assembled into 43,753 high confidence transcript contigs. In excess of 50% of the assembled transcripts were annotated with domain- or protein sequence similarity derived functional information. The reference adult P. cervi transcriptome will serve as a basis for future work on the biology of this important parasite. Using the widely investigated trematode virulence factor and vaccine candidate Cathepsin L as an example, the epitope GPISIAINA was found to be conserved in P. cervi isolated from three different host species supporting its candidacy for vaccine development and illustrating the utility of the adult P. cervi transcriptome. PMID:26049095

Adventitious rooting is the most important mechanism underlying vegetative propagation and an important strategy for plant propagation under environmental stress. The present study was conducted to obtain transcriptomic data and examine gene expression using RNA-Seq and bioinformatics analysis, thereby providing a foundation for understanding the molecular mechanisms controlling adventitious rooting. Three cDNA libraries constructed from mRNA samples from mung bean hypocotyls during adventitious rooting were sequenced. These three samples generated a total of 73 million, 60 million, and 59 million 100-bp reads, respectively. These reads were assembled into 78,697 unigenes with an average length of 832 bp, totaling 65 Mb. The unigenes were aligned against six public protein databases, and 29,029 unigenes (36.77%) were annotated using BLASTx. Among them, 28,225 (35.75%) and 28,119 (35.62%) unigenes had homologs in the TrEMBL and NCBI non-redundant (Nr) databases, respectively. Of these unigenes, 21,140 were assigned to gene ontology classes, and a total of 11,990 unigenes were classified into 25 KOG functional categories. A total of 7,357 unigenes were annotated to 4,524 KOs, and 4,651 unigenes were mapped onto 342 KEGG pathways using BLAST comparison against the KEGG database. A total of 11,717 unigenes were differentially expressed (fold change>2) during the root induction stage, with 8,772 unigenes down-regulated and 2,945 unigenes up-regulated. A total of 12,737 unigenes were differentially expressed during the root initiation stage, with 9,303 unigenes down-regulated and 3,434 unigenes up-regulated. A total of 5,334 unigenes were differentially expressed between the root induction and initiation stage, with 2,167 unigenes down-regulated and 3,167 unigenes up-regulated. qRT-PCR validation of the 39 genes with known functions indicated a strong correlation (92.3%) with the RNA-Seq data. The GO enrichment, pathway mapping, and gene expression profiles reveal

Background Simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low. Methodology/Principal Findings Illumina paired-end sequencing technology produced over 53 million sequencing reads from C. nankingense mRNA. The subsequent de novo assembly yielded 70,895 unigenes, of which 45,789 (64.59%) unigenes showed similarity to the sequences in NCBI database. Out of 45,789 sequences, 107 have hits to the Chrysanthemum Nr protein database; 679 and 277 sequences have hits to the database of Helianthus and Lactuca species, respectively. MISA software identified a large number of putative EST-SSRs, allowing 1,788 primer pairs to be designed from the de novo transcriptome sequence and a further 363 from archival EST sequence. Among 100 primer pairs randomly chosen, 81 markers have amplicons and 20 are polymorphic for genotypes analysis in Chrysanthemum. The results showed that most (but not all) of the assays were transferable across species and that they exposed a significant amount of allelic diversity. Conclusions/Significance SSR markers acquired by transcriptome sequencing are potentially useful for marker-assisted breeding and genetic analysis in the genus Chrysanthemum and its related genera. PMID:23626799

The microgravity environment of spaceflight expeditions has been associated with altered microbial responses. This study explores the characterization of Serratia marcescensis grown in a spaceflight environment at the phenotypic, transcriptomic and proteomic levels. From November 1, 2011 to November 17, 2011, a strain of S. marcescensis was sent into space for 398 h on the Shenzhou VIII spacecraft, and ground simulation was performed as a control (LCT-SM213). After the flight, two mutant strains (LCT-SM166 and LCT-SM262) were selected for further analysis. Although no changes in the morphology, post-culture growth kinetics, hemolysis or antibiotic sensitivity were observed, the two mutant strains exhibited significant changes in their metabolic profiles after exposure to spaceflight. Enrichment analysis of the transcriptome showed that the differentially expressed genes of the two spaceflight strains and the ground control strain mainly included those involved in metabolism and degradation. The proteome revealed that changes at the protein level were also associated with metabolic functions, such as glycolysis/gluconeogenesis, pyruvate metabolism, arginine and proline metabolism and the degradation of valine, leucine and isoleucine. In summary S. marcescens showed alterations primarily in genes and proteins that were associated with metabolism under spaceflight conditions, which gave us valuable clues for future research.

Integration of multiple profiling data and construction of functional gene networks may provide additional insights into the molecular mechanisms of complex diseases. Osteoporosis is a worldwide public health problem, but the complex gene-gene interactions, post-transcriptional modifications and regulation of functional networks are still unclear. To gain a comprehensive understanding of osteoporosis etiology, transcriptome gene expression microarray, epigenomic miRNA microarray and methylome sequencing were performed simultaneously in 5 high hip BMD (Bone Mineral Density) subjects and 5 low hip BMD subjects. SPIA (Signaling Pathway Impact Analysis) and PCST (Prize Collecting Steiner Tree) algorithm were used to perform pathway-enrichment analysis and construct the interaction networks. Through integrating the transcriptomic and epigenomic data, firstly we identified 3 genes (FAM50A, ZNF473 and TMEM55B) and one miRNA (hsa-mir-4291) which showed the consistent association evidence from both gene expression and methylation data; secondly in network analysis we identified an interaction network module with 12 genes and 11 miRNAs including AKT1, STAT3, STAT5A, FLT3, hsa-mir-141 and hsa-mir-34a which have been associated with BMD in previous studies. This module revealed the crosstalk among miRNAs, mRNAs and DNA methylation and showed four potential regulatory patterns of gene expression to influence the BMD status. In conclusion, the integration of multiple layers of omics can yield in-depth results than analysis of individual omics data respectively. Integrative analysis from transcriptomics and epigenomic data improves our ability to identify causal genetic factors, and more importantly uncover functional regulation pattern of multi-omics for osteoporosis etiology. PMID:26390436

A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation. DOI: http://dx.doi.org/10.7554/eLife.02510.001 PMID:24867642

Research on tests for scale equality have focused exclusively on an overall test statistic and have not examined procedures for identifying specific differences in multiple group designs. The present study compares four contrast analysis procedures for scale differences in the single factor four-group design: (1) Tukey HSD; (2) Kramer-Tukey; (3)…

This article describes a follow-up analysis of the Spiritual Competency Scale, which initially validated ASERVIC's (Association for Spiritual, Ethical and Religious Values in Counseling) spiritual competencies. The study examined whether the factor structure of the Spiritual Competency Scale would be supported by participants (i.e., ASERVIC…

In this work, the effect of rosemary extracts rich on polyphenols obtained using pressurized fluids was investigated on the gene expression of human SW480 and HT29 colon cancer cells. The application of transcriptomic profiling and functional enrichment analysis was done via two computational approaches, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis. These two approaches were used for functional enrichment analysis as a previous step for a reliable interpretation of the data obtained from microarray analysis. Reverse transcription quantitative-PCR was used to confirm relative changes in mRNA levels of selected genes from microarrays. The selection of genes was based on their expression change, adjusted p value, and known biological function. According to genome-wide transcriptomicsanalysis, rosemary polyphenols altered the expression of ~4 % of the genes covered by the Affymetrix Human Gene 1.0ST chip in both colon cancer cells. However, only ~18 % of the differentially expressed genes were common to both cell lines, indicating markedly different expression profiles in response to the treatment. Differences in induction of G2/M arrest observed by rosemary polyphenols in the two colon adenocarcinoma cell lines suggest that the extract may be differentially effective against tumors with specific mutational pattern. From our results, it is also concluded that rosemary polyphenols induced a low degree of apoptosis indicating that other multiple signaling pathways may contribute to colon cancer cell death. PMID:22923011

In nature plants are often simultaneously challenged by different biotic and abiotic stresses. Although the mechanisms underlying plant responses against single stress have been studied considerably, plant tolerance mechanisms under combined stress is not understood. Also, the mechanism used to combat independently and sequentially occurring many number of biotic and abiotic stresses has also not systematically studied. From this context, in this study, we attempted to explore the shared response of sunflower plants to many independent stresses by using meta-analysis of publically available transcriptome data and transcript profiling by quantitative PCR. Further, we have also analyzed the possible role of the genes so identified in contributing to combined stress tolerance. Meta-analysis of transcriptomic data from many abiotic and biotic stresses indicated the common representation of oxidative stress responsive genes. Further, menadione-mediated oxidative stress in sunflower seedlings showed similar pattern of changes in the oxidative stress related genes. Based on this a large scale screening of 55 sunflower genotypes was performed under menadione stress and those contrasting in oxidative stress tolerance were identified. Further to confirm the role of genes identified in individual and combined stress tolerance the contrasting genotypes were individually and simultaneously challenged with few abiotic and biotic stresses. The tolerant hybrid showed reduced levels of stress damage both under combined stress and few independent stresses. Transcript profiling of the genes identified from meta-analysis in the tolerant hybrid also indicated that the selected genes were up-regulated under individual and combined stresses. Our results indicate that menadione-based screening can identify genotypes not only tolerant to multiple number of individual biotic and abiotic stresses, but also the combined stresses. PMID:27314499

Background The diamondback moth Plutella xyllostella has developed a high level of resistance to the latest insecticide chlorantraniliprole. A better understanding of P. xylostella’s resistance mechanism to chlorantraniliprole is needed to develop effective approaches for insecticide resistance management. Principal Findings To provide a comprehensive insight into the resistance mechanisms of P. xylostella to chlorantraniliprole, transcriptome assembly and tag-based digital gene expression (DGE) system were performed using Illumina HiSeq™ 2000. The transcriptomeanalysis of the susceptible strain (SS) provided 45,231 unigenes (with the size ranging from 200 bp to 13,799 bp), which would be efficient for analyzing the differences in different chlorantraniliprole-resistant P. xylostella stains. DGE analysis indicated that a total of 1215 genes (189 up-regulated and 1026 down-regulated) were gradient differentially expressed among the susceptible strain (SS) and different chlorantraniliprole-resistant P. xylostella strains, including low-level resistance (GXA), moderate resistance (LZA) and high resistance strains (HZA). A detailed analysis of gradient differentially expressed genes elucidated the existence of a phase-dependent divergence of biological investment at the molecular level. The genes related to insecticide resistance, such as P450, GST, the ryanodine receptor, and connectin, had different expression profiles in the different chlorantraniliprole-resistant DGE libraries, suggesting that the genes related to insecticide resistance are involved in P. xylostella resistance development against chlorantraniliprole. To confirm the results from the DGE, the expressional profiles of 4 genes related to insecticide resistance were further validated by qRT-PCR analysis. Conclusions The obtained transcriptome information provides large gene resources available for further studying the resistance development of P. xylostella to pesticides. The DGE data provide

Transcriptomeanalysis of bovine mammary development has provided insight into regulation of mammogenesis. However, previous studies primarily examined expression of epithelial and stromal tissues combined, and consequently did not account for tissue specific contribution to mammary development. Our objective was to identify differences in gene expression in epithelial and intralobular stromal compartments. Tissue was biopsied from non-lactating dairy cows 3 weeks prepartum, cut into explants and incubated for 2 hr with insulin and hydrocortisone. Epithelial and intralobular stromal tissues were isolated with laser capture microdissection. Global gene expression was measured with Bovine Affymetrix GeneChips, and data were preprocessed using RMA method. Moderated t-tests from gene-specific linear model analysis with cell type as a fixed effect showed more than 3,000 genes were differentially expressed between tissues (P<0.05; FDR<0.17). Analysis of epithelial and stromal transcriptomes using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathways Analysis (IPA) showed that epithelial and stromal cells contributed distinct molecular signatures. Epithelial signatures were enriched with gene sets for protein synthesis, metabolism and secretion. Stromal signatures were enriched with genes that encoded molecules important to signaling, extracellular matrix composition and remodeling. Transcriptome differences also showed evidence for paracrine interactions between tissues in stimulation of IGF1 signaling pathway, stromal reaction, angiogenesis, neurogenesis, and immune response. Molecular signatures point to the dynamic role the stroma plays in prepartum mammogenesis and highlight the importance of examining the roles of cell types within the mammary gland when targeting therapies and studying mechanisms that affect milk production. PMID:21829467

Stainless steel, polymethylmethacrylate and polytetrafluoroethylene coupons were analysed for surface topographical and adhesion force characteristics using tapping mode atomic force microscopy and force-distance microscopy techniques. The two polymer materials were surface modified by polishing with silicon carbide papers of known grade. The struvite scaling rate was determined for each coupon and related to the data gained from the surface analysis. The scaling rate correlated well with adhesion force measurements indicating that lower energy materials scale at a lower rate. The techniques outlined in the paper provide a method for the rapid screening of materials in potential scaling applications. PMID:14982180

Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV). There are no effective vaccines or treatment available, and the virus virulence determinants and pathogenesis are not fully understood. Here, we describe the sequencing of RNA extracted from Crandell Rees Feline Kidney (CRFK) cells infected with FIPV using the Illumina next-generation sequencing approach. Bioinformatics analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench is used to map both control and infected cells. Kal's Z test statistical analysis is used to analyze the differentially expressed genes from the infected CRFK cells. In addition, RT-qPCR analysis is used for further transcriptional profiling of selected genes in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diagnosed cats. PMID:25720485

Background Abiotic stress, including low temperature, limits the productivity and geographical distribution of plants, which has led to significant interest in understanding the complex processes that allow plants to adapt to such stresses. The wide range of physiological, biochemical and molecular changes that occur in plants exposed to low temperature require a robust global approach to studying the response. We have employed Serial Analysis of Gene Expression (SAGE) to uncover changes in the transcriptome of Arabidopsis thaliana over a time course of low temperature stress. Results Five SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4°C). Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance. Interpretation of these data was facilitated by representing the SAGE data by gene identifier, allowing more robust statistical analysis, cross-platform comparisons and the identification of genes sharing common expression profiles. Simultaneous statistical calculations across all five libraries identified 920 low temperature responsive genes, only 24% of which overlapped with previous global expression analysis performed using microarrays, although similar functional categories were affected. Clustering of the differentially regulated genes facilitated the identification of novel loci correlated with the development of freezing tolerance. Analysis of their promoter sequences revealed subsets of genes that were independent of CBF and ABA regulation and could provide a mechanism for elucidating complementary signalling pathways. The SAGE data emphasised the complexity of the plant response, with alternate pre-mRNA processing events increasing at low temperatures and antisense transcription

Ginger (Zingiber officinale Rosc.), a well-known member of family Zingiberaceae, is bestowed with number of medicinal properties which is because of the secondary metabolites, essential oil and oleoresin, it contains in its rhizome. The drug yielding potential is known to depend on agro-climatic conditions prevailing at the place cultivation. Present study deals with comparative transcriptomeanalysis of two sample of elite ginger variety Suprabha collected from two different agro-climatic zones of Odisha. Transcriptome assembly for both the samples was done using next generation sequencing methodology. The raw data of size 10.8 and 11.8 GB obtained from analysis of two rhizomes S1Z4 and S2Z5 collected from Bhubaneswar and Koraput and are available in NCBI accession number SAMN03761169 and SAMN03761176 respectively. We identified 60,452 and 54,748 transcripts using trinity tool respectively from ginger rhizome of S1Z4 and S2Z5. The transcript length varied from 300 bp to 15,213 bp and 8988 bp and N50 value of 1415 bp and 1334 bp respectively for S1Z4 and S2Z5. To the best of our knowledge, this is the first comparative transcriptomeanalysis of elite ginger cultivars Suprabha from two different agro-climatic conditions of Odisha, India which will help to understand the effect of agro-climatic conditions on differential expression of secondary metabolites. PMID:27408809

Cytokinin plant hormones have been shown to play an important role in plant response to abiotic stresses. Herein, we expand upon the findings of Pospíšilová et al. [30] regarding preparation of novel transgenic barley lines overexpressing cytokinin dehydrogenase 1 gene from Arabidopsis under the control of mild root-specific promotor of maize β-glycosidase. These lines showed drought-tolerant phenotype mainly due to alteration of root architecture and stronger lignification of root tissue. A detailed transcriptomicanalysis of roots of transgenic plants subjected to revitalization after drought stress revealed attenuated response through the HvHK3 cytokinin receptor and up-regulation of two transcription factors implicated in stress responses and abscisic acid sensitivity. Increased expression of several genes involved in the phenylpropanoid pathway as well as of genes encoding arogenate dehydratase/lyase participating in phenylalanine synthesis was found in roots during revitalization. Although more precursors of lignin synthesis were present in roots after drought stress, final lignin accumulation did not change compared to that in plants grown under optimal conditions. Changes in transcriptome indicated a higher auxin turnover in transgenic roots. The same analysis in leaves revealed that genes encoding putative enzymes responsible for production of jasmonates and other volatile compounds were up-regulated. Although transgenic barley leaves showed lower chlorophyll content and down-regulation of genes encoding proteins involved in photosynthesis than did wild-type plants when cultivated under optimal conditions, they did show a tendency to return to initial photochemical activities faster than did wild-type leaves when re-watered after severe drought stress. In contrast to optimal conditions, comparative transcriptomicanalysis of revitalized leaves displayed up-regulation of genes encoding enzymes and proteins involved in photosynthesis, and especially

Proteomics analysis of paired cancer and control tissue can be applied to investigate pathological processes in tumors. Advancements in data-independent acquisition mass spectrometry allow for highly reproducible quantitative analysis of complex proteomic patterns. Optimized sample preparation workflows enable integrative multi-omics studies from the same tissue specimens. We performed ion mobility enhanced, data-independent acquisition MS to characterize the proteome of 21 lung tumor tissues including adenocarcinoma and squamous cell carcinoma (SCC) as compared to control lung tissues of the same patient each. Transcriptomic data were generated for the same specimens. The quantitative proteomic patterns and mRNA abundances were subsequently analyzed using systems biology approaches. We report a significantly (p = 0.0001) larger repertoire of proteins in cancer tissues. 12 proteins were higher in all tumor tissues as compared to matching control tissues. Three proteins, CAV1, CAV2, and RAGE, were vice versa higher in all controls. We also identified characteristic SCC and adenocarcinoma protein patterns. Principal Component Analysis provided evidence that not only cancer from control tissue but also tissue from adenocarcinoma and SCC can be differentiated. Transcriptomic levels of key proteins measured from the same matched tissue samples correlated with the observed protein patterns. The applied study set-up with paired lung tissue specimens of which different omics are measured, is generally suited for an integrated multi-omics analysis. PMID:26930711

We present Oqtans, an open-source workbench for quantitative transcriptomeanalysis, that is integrated in Galaxy. Its distinguishing features include customizable computational workflows and a modular pipeline architecture that facilitates comparative assessment of tool and data quality. Oqtans integrates an assortment of machine learning-powered tools into Galaxy, which show superior or equal performance to state-of-the-art tools. Implemented tools comprise a complete transcriptomeanalysis workflow: short-read alignment, transcript identification/quantification and differential expression analysis. Oqtans and Galaxy facilitate persistent storage, data exchange and documentation of intermediate results and analysis workflows. We illustrate how Oqtans aids the interpretation of data from different experiments in easy to understand use cases. Users can easily create their own workflows and extend Oqtans by integrating specific tools. Oqtans is available as (i) a cloud machine image with a demo instance at cloud.oqtans.org, (ii) a public Galaxy instance at galaxy.cbio.mskcc.org, (iii) a git repository containing all installed software (oqtans.org/git); most of which is also available from (iv) the Galaxy Toolshed and (v) a share string to use along with Galaxy CloudMan. PMID:24413671

Background Dormancy of buds is a critical developmental process that allows perennial plants to survive extreme seasonal variations in climate. Dormancy transitions in underground crown buds of the model herbaceous perennial weed leafy spurge were investigated using a 23 K element cDNA microarray. These data represent the first large-scaletranscriptomeanalysis of dormancy in underground buds of an herbaceous perennial species. Crown buds collected monthly from August through December, over a five year period, were used to monitor the changes in the transcriptome during dormancy transitions. Results Nearly 1,000 genes were differentially-expressed through seasonal dormancy transitions. Expected patterns of gene expression were observed for previously characterized genes and physiological processes indicated that resolution in our analysis was sufficient for identifying shifts in global gene expression. Conclusion Gene ontology of differentially-expressed genes suggests dormancy transitions require specific alterations in transport functions (including induction of a series of mitochondrial substrate carriers, and sugar transporters), ethylene, jasmonic acid, auxin, gibberellic acid, and abscisic acid responses, and responses to stress (primarily oxidative and cold/drought). Comparison to other dormancy microarray studies indicated that nearly half of the genes identified in our study were also differentially expressed in at least two other plant species during dormancy transitions. This comparison allowed us to identify a particular MADS-box transcription factor related to the DORMANCY ASSOCIATED MADS-BOX genes from peach and hypothesize that it may play a direct role in dormancy induction and maintenance through regulation of FLOWERING LOCUS T. PMID:19014493

Quinclorac is a highly selective auxin-type herbicide and is widely used in the effective control of barnyard grass in paddy rice fields, improving the world's rice yield. The herbicide mode of action of quinclorac has been proposed, and hormone interactions affecting quinclorac signaling has been identified. Because of widespread use, quinclorac may be transported outside rice fields with the drainage waters, leading to soil and water pollution and other environmental health problems. In this study, we used 57K Affymetrix rice whole-genome array to identify quinclorac signaling response genes to study the molecular mechanisms of action and detoxification of quinclorac in rice plants. Overall, 637 probe sets were identified with differential expression levels under either 6 or 24 h of quinclorac treatment. Auxin-related genes such as GH3 and OsIAAs responded to quinclorac treatment. Gene Ontology analysis showed that genes of detoxification-related family genes were significantly enriched, including cytochrome P450, GST, UGT, and ABC and drug transporter genes. Moreover, real-time RT-PCR analysis showed that top candidate genes of P450 families such as CYP81, CYP709C, and CYP72A were universally induced by different herbicides. Some Arabidopsis genes of the same P450 family were up-regulated under quinclorac treatment. We conducted rice whole-genome GeneChip analysis and the first global identification of quinclorac response genes. This work may provide potential markers for detoxification of quinclorac and biomonitors of environmental chemical pollution. PMID:26483837

Analysis of gene expression has contributed to a plethora of biological and medical research studies. Microarrays have been intensively used for the profiling of gene expression during diverse developmental processes, treatments and diseases. New massively parallel sequencing methods, often named as RNA-sequencing (RNA-seq) are extensively improving our understanding of gene regulation and signaling networks. Computational methods developed originally for microarrays analysis can now be optimized and applied to genome-wide studies in order to have access to a better comprehension of the whole transcriptome. This review addresses current challenges on RNA-seq analysis and specifically focuses on new bioinformatics tools developed for time series experiments. Furthermore, possible improvements in analysis, data integration as well as future applications of differential expression analysis are discussed. PMID:26430493

Analysis of gene expression has contributed to a plethora of biological and medical research studies. Microarrays have been intensively used for the profiling of gene expression during diverse developmental processes, treatments and diseases. New massively parallel sequencing methods, often named as RNA-sequencing (RNA-seq) are extensively improving our understanding of gene regulation and signaling networks. Computational methods developed originally for microarrays analysis can now be optimized and applied to genome-wide studies in order to have access to a better comprehension of the whole transcriptome. This review addresses current challenges on RNA-seq analysis and specifically focuses on new bioinformatics tools developed for time series experiments. Furthermore, possible improvements in analysis, data integration as well as future applications of differential expression analysis are discussed. PMID:26430493

The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host-pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host-pathogen interactions in infection models. PMID:27442864

The aim of this study was to provide comprehensive insights into the genetic background of sturgeon by transcriptome study. We performed a de novo assembly of the Amur sturgeon Acipenser schrenckii transcriptome using Illumina Hiseq 2000 sequencing. A total of 148,817 non-redundant unigenes with base length of approximately 121,698,536 bp and ranges from 201 to 26,789 bp were obtained. All the unigenes were classified into 3368 distinct categories and 145,449 singletons by homologous transcript cluster analysis. In all, 46,865 (31.49%) unigenes showed homologous matches with Nr database and 32,214 (21.65%) unigenes were matched to Nt database. In total, 24,862 unigenes were categorized into significantly enriched 52 function groups by GO analysis, and 38,436 unigenes were classified into 25 groups by KOG prediction, as well as 128 enriched KEGG pathways were identified by 45,598 unigenes (P < 0.05). Subsequently, a total of 19,860 SSRs markers were identified with the abundant di-nucleotide type (10,658; 53.67%) and the most AT/TA motif repeats (2689; 13.54%). A total of 1341 conserved lncRNAs were identified by a customized pipeline. Our study provides new sequence and function information for A. schrenckii, which will be the basis for further genetic studies on sturgeon species. The huge number of potential SSRs and putatively conserved lncRNAs isolated by the transcriptome also shed light on research in many fields, including the evolution, conservation management, and biological processes in sturgeon. PMID:27420941

We report the first combined analysis of whole-genome sequence, detailed clinical history, and transcriptome sequence of multiple prostate cancer metastases in a single patient (A21). Whole-genome and transcriptome sequence was obtained from nine anatomically separate metastases, and targeted DNA sequencing was performed in cancerous and noncancerous foci within the primary tumor specimen removed 5 yr before death. Transcriptomeanalysis revealed increased expression of androgen receptor (AR)-regulated genes in liver metastases that harbored an AR p.L702H mutation, suggesting a dominant effect by the mutation despite being present in only one of an estimated 16 copies per cell. The metastases harbored several alterations to the PI3K/AKT pathway, including a clonal truncal mutation in PIK3CG and present in all metastatic sites studied. The list of truncal genomic alterations shared by all metastases included homozygous deletion of TP53, hemizygous deletion of RB1 and CHD1, and amplification of FGFR1. If the patient were treated today, given this knowledge, the use of second-generation androgen-directed therapies, cessation of glucocorticoid administration, and therapeutic inhibition of the PI3K/AKT pathway or FGFR1 receptor could provide personalized benefit. Three previously unreported truncal clonal missense mutations (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) were expressed at the RNA level and assessed as druggable. The truncal status of mutations may be critical for effective actionability and merit further study. Our findings suggest that a large set of deeply analyzed cases could serve as a powerful guide to more effective prostate cancer basic science and personalized cancer medicine clinical trials. PMID:27148588

Globally, a chronic hepatitis B virus (HBV) infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication. PMID:26891448

We report the first combined analysis of whole-genome sequence, detailed clinical history, and transcriptome sequence of multiple prostate cancer metastases in a single patient (A21). Whole-genome and transcriptome sequence was obtained from nine anatomically separate metastases, and targeted DNA sequencing was performed in cancerous and noncancerous foci within the primary tumor specimen removed 5 yr before death. Transcriptomeanalysis revealed increased expression of androgen receptor (AR)-regulated genes in liver metastases that harbored an AR p.L702H mutation, suggesting a dominant effect by the mutation despite being present in only one of an estimated 16 copies per cell. The metastases harbored several alterations to the PI3K/AKT pathway, including a clonal truncal mutation in PIK3CG and present in all metastatic sites studied. The list of truncal genomic alterations shared by all metastases included homozygous deletion of TP53, hemizygous deletion of RB1 and CHD1, and amplification of FGFR1. If the patient were treated today, given this knowledge, the use of second-generation androgen-directed therapies, cessation of glucocorticoid administration, and therapeutic inhibition of the PI3K/AKT pathway or FGFR1 receptor could provide personalized benefit. Three previously unreported truncal clonal missense mutations (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) were expressed at the RNA level and assessed as druggable. The truncal status of mutations may be critical for effective actionability and merit further study. Our findings suggest that a large set of deeply analyzed cases could serve as a powerful guide to more effective prostate cancer basic science and personalized cancer medicine clinical trials. PMID:27148588

Background Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development. Results The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat. Conclusions This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species. PMID:23861841

BACKGROUND: Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development. RESULTS: The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat. CONCLUSIONS: This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species.

Esophageal cancer is often diagnosed at an advanced stage. Diagnostic markers are needed for achieving a cure in esophageal cancer detecting and treating tumor cells earlier. In patients with locally advanced squamous cell carcinoma of the esophagus (ESCC), we profiled the gene expression of ESCC compared to corresponding normal biopsies for diagnostic markers by genome microarrays. Profiling of gene expression identified 4844 genes differentially expressed, 2122 upregulated and 2722 downregulated in ESCC. Twenty-three overexpressed candidates with best scores from significance analysis have been selected for further analysis by TaqMan low-density array-technique using a validation cohort of 40 patients. The verification rate was 100 % for ESCC. Twenty-two markers were additionally overexpressed in adenocarcinoma of the esophagus (EAC). The markers significantly overexpressed already in earlier tumor stages (pT1-2) of both histological subtypes (n = 19) have been clustered in a "diagnostic signature": PLA2G7, PRAME, MMP1, MMP3, MMP12, LIlRB2, TREM2, CHST2, IGFBP2, IGFBP7, KCNJ8, EMILIN2, CTHRC1, EMR2, WDR72, LPCAT1, COL4A2, CCL4, and SNX10. The marker signature will be translated to clinical practice to prove its diagnostic impact. This diagnostic signature may contribute to the earlier detection of tumor cells, with the aim to complement clinical techniques resulting in the development of better detection of concepts of esophageal cancer for earlier therapy and more favorite prognosis. PMID:26631031

Background Amaranthus hypochondriacus, a grain amaranth, is a C4 plant noted by its ability to tolerate stressful conditions and produce highly nutritious seeds. These possess an optimal amino acid balance and constitute a rich source of health-promoting peptides. Although several recent studies, mostly involving subtractive hybridization strategies, have contributed to increase the relatively low number of grain amaranth expressed sequence tags (ESTs), transcriptomic information of this species remains limited, particularly regarding tissue-specific and biotic stress-related genes. Thus, a large scaletranscriptomeanalysis was performed to generate stem- and (a)biotic stress-responsive gene expression profiles in grain amaranth. Results A total of 2,700,168 raw reads were obtained from six 454 pyrosequencing runs, which were assembled into 21,207 high quality sequences (20,408 isotigs + 799 contigs). The average sequence length was 1,064 bp and 930 bp for isotigs and contigs, respectively. Only 5,113 singletons were recovered after quality control. Contigs/isotigs were further incorporated into 15,667 isogroups. All unique sequences were queried against the nr, TAIR, UniRef100, UniRef50 and Amaranthaceae EST databases for annotation. Functional GO annotation was performed with all contigs/isotigs that produced significant hits with the TAIR database. Only 8,260 sequences were found to be homologous when the transcriptomes of A. tuberculatus and A. hypochondriacus were compared, most of which were associated with basic house-keeping processes. Digital expression analysis identified 1,971 differentially expressed genes in response to at least one of four stress treatments tested. These included several multiple-stress-inducible genes that could represent potential candidates for use in the engineering of stress-resistant plants. The transcriptomic data generated from pigmented stems shared similarity with findings reported in developing stems of Arabidopsis and

Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis. PMID:27064558

Tulipa edulis (Miq.) Baker is an important medicinal plant with a variety of anti-cancer properties. The stolon is one of the main asexual reproductive organs of T. edulis and possesses a unique morphology. To explore the molecular mechanism of stolon formation, we performed an RNA-seq analysis of the transcriptomes of stolons at three developmental stages. In the present study, 15.49 Gb of raw data were generated and assembled into 74,006 unigenes, and a total of 2,811 simple sequence repeats were detected in T. edulis. Among the three libraries of stolons at different developmental stages, there were 5,119 differentially expressed genes (DEGs). A functional annotation analysis based on sequence similarity queries of the GO, COG, KEGG databases showed that these DEGs were mainly involved in many physiological and biochemical processes, such as material and energy metabolism, hormone signaling, cell growth, and transcription regulation. In addition, quantitative real-time PCR analysis revealed that the expression patterns of the DEGs were consistent with the transcriptome data, which further supported a role for the DEGs in stolon formation. This study provides novel resources for future genetic and molecular studies in T. edulis. PMID:27064558

The analysis of transcriptomics data is able to give an overview of cellular processes, but requires sophisticated bioinformatics tools and methods to identify the changes. Pathway analysis software, like PathVisio, captures the information about biological pathways from databases and brings this together with the experimental data to enable visualization and understanding of the underlying processes. Rett syndrome is a rare disease, but still one of the most abundant causes of intellectual disability in females. Cause of this neurological disorder is mutation of one single gene, the methyl-CpG-binding protein 2 (MECP2) gene. This gene is responsible for many steps in neuronal development and function. Although the genetic mutation and the clinical phenotype are well described, the molecular pathways linking them are not yet fully elucidated. In this study we demonstrate a workflow for the analysis of transcriptomics data to identify biological pathways and processes which are changed in a Mecp2 (-/y) mouse model. PMID:27517371

The biosynthesis of chlorophyll and other tetrapyrroles is a vital but poorly understood process. Recent genomic advances with the unicellular green algae Chlamydomonas reinhardtii have created opportunity to more closely examine the mechanisms of the chlorophyll biosynthesis pathway via transcriptomeanalysis. Manganese is a nutrient of interest for complex reactions because of its multiple stable oxidation states and role in molecular oxygen coordination. C. reinhardtii was cultured in Manganese-deplete Tris-acetate-phosphate (TAP) media for 24 hours and used to create cDNA libraries for sequencing using Illumina TruSeq technology. Transcriptomeanalysis provided intriguing insight on possible regulatory mechanisms in the pathway. Evidence supports similarities of GTR (Glutamyl-tRNA synthase) to its Chlorella vulgaris homolog in terms of Mn requirements. Data was also suggestive of Mn-related compensatory up-regulation for pathway proteins CHLH1 (Manganese Chelatase), GUN4 (Magnesium chelatase activating protein), and POR1 (Light-dependent protochlorophyllide reductase). Intriguingly, data suggests possible reciprocal expression of oxygen dependent CPX1 (coproporphyrinogen III oxidase) and oxygen independent CPX2. Further analysis using RT-PCR could provide compelling evidence for several novel regulatory mechanisms in the chlorophyll biosynthesis pathway.

Red Palm Weevil (RPW, Rhynchophorus ferrugineus Olivier) threatens most palm species worldwide. Until now, no studies have analyzed the gene regulatory networks of Phoenix canariensis (Chabaud) in response to RPW attacks. The aim of this study was to fill this knowledge gap. Providing this basic knowledge is very important to improve its management. Results: A deep transcriptomeanalysis was performed on fully expanded leaves of healthy non-infested trees and attacked trees at two symptom stages (middle and late infestation). A total of 54 genes were significantly regulated during middle stage. Pathway enrichment analysis showed that phenylpropanoid-related pathways were induced at this stage. More than 3300 genes were affected during late stage of attacks. Higher transcript abundances were observed for lipid fatty acid metabolism (fatty acid and glycerolipids), tryptophan metabolism, phenylpropanoid metabolism. Key RPW-modulated genes involved in innate response mediated by hormone crosstalk were observed belonging to auxin, jasmonate and salicylic acid (SA) pathways. Among transcription factors, some WRKYs were clearly induced. qRT-PCR validation confirmed the upregulation of key genes chosen as validation of transcriptomicanalysis. Conclusion: A subset of these genes may be further analyzed in future studies to confirm their specificity to be induced by RPW infestations. PMID:26528297

Perfluorooctane sulfonate (PFOS), a hepato-toxicant and potential non-genotoxic carcinogen, was widely used in industrial and commercial products. Recent studies have revealed the ubiquitous occurrence of PFOS in the environment and in humans worldwide. The widespread contamination of PFOS in human serum raised concerns about its long-term toxic effects and its potential risks to human health. Using fatty liver mutant foie gras (fgr(-/-))/transport protein particle complex 11 (trappc11(-/-)) and PFOS-exposed wild-type zebrafish embryos as the study model, together with RNA sequencing and comparative transcriptomicanalysis, we identified 499 and 1414 differential expressed genes (DEGs) in PFOS-exposed wild-type and trappc11 mutant zebrafish, respectively. Also, the gene ontology analysis on common deregulated genes was found to be associated with different metabolic processes such as the carbohydrate metabolic process, glycerol ether metabolic process, mannose biosynthetic process, de novo' (Guanosine diphosphate) GDP-l-fucose biosynthetic process, GDP-mannose metabolic process and galactose metabolic process. Ingenuity Pathway Analysis further highlighted that these deregulated gene clusters are closely related to hepatitis, inflammation, fibrosis and cirrhosis of liver cells, suggesting that PFOS can cause liver pathogenesis and non-alcoholic fatty liver disease in zebrafish. The transcriptomic alterations revealed may serve as biomarkers for the hepatotoxic effect of PFOS. PMID:27289203

Virus infection of plants may induce a variety of disease symptoms. However, little is known about the molecular mechanism of systemic symptom development in infected plants. Here we performed the first next-generation sequencing study to identify gene expression changes associated with disease development in tobacco plants (Nicotiana tabacum cv. Xanthi nc) induced by infection with the M strain of Cucumber mosaic virus (M-CMV). Analysis of the tobacco transcriptome by RNA-Seq identified 95,916 unigenes, 34,408 of which were new transcripts by database searches. Deep sequencing was subsequently used to compare the digital gene expression (DGE) profiles of the healthy plants with the infected plants at six sequential disease development stages, including vein clearing, mosaic, severe chlorosis, partial and complete recovery, and secondary mosaic. Thousands of differentially expressed genes were identified, and KEGG pathway analysis of these genes suggested that many biological processes, such as photosynthesis, pigment metabolism and plant-pathogen interaction, were involved in systemic symptom development. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in virus-infected plants. This information will help further our understanding of the detailed mechanisms of plant responses to viral infection. PMID:22952684

Licorice is an important traditional Chinese medicine with clinical and industrial applications. Genetic resources of licorice are insufficient for analysis of molecular biology and genetic functions; as such, transcriptome sequencing must be conducted for functional characterization and development of molecular markers. In this study, transcriptome sequencing on the Illumina HiSeq 2500 sequencing platform generated a total of 5.41 Gb clean data. De novo assembly yielded a total of 46,641 unigenes. Comparison analysis using BLAST showed that the annotations of 29,614 unigenes were conserved. Further study revealed 773 genes related to biosynthesis of secondary metabolites of licorice, 40 genes involved in biosynthesis of the terpenoid backbone, and 16 genes associated with biosynthesis of glycyrrhizic acid. Analysis of unigenes larger than 1 Kb with a length of 11,702 nt presented 7,032 simple sequence repeats (SSR). Sixty-four of 69 randomly designed and synthesized SSR pairs were successfully amplified, 33 pairs of primers were polymorphism in in Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., Glycyrrhiza glabra L. and Glycyrrhiza pallidiflora Maxim. This study not only presents the molecular biology data of licorice but also provides a basis for genetic diversity research and molecular marker-assisted breeding of licorice. PMID:26571372

Domesticated species occupy a special place in the human world due to their economic and cultural value. In the era of genomic research, domesticated species provide unique advantages for investigation of diseases and complex phenotypes. RNA sequencing, or RNA-seq, has recently emerged as a new approach for studying transcriptional activity of the whole genome, changing the focus from individual genes to gene networks. RNA-seq analysis in domesticated species may complement genome-wide association studies of complex traits with economic importance or direct relevance to biomedical research. However, RNA-seq studies are more challenging in domesticated species than in model organisms. These challenges are at least in part associated with the lack of quality genome assemblies for some domesticated species and the absence of genome assemblies for others. In this review, we discuss strategies for analyzing RNA-seq data, focusing particularly on questions and examples relevant to domesticated species. PMID:26917953

Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed ‘signature’ genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases. PMID:20360305

Background Heliconius butterfly wing pattern diversity offers a unique opportunity to investigate how natural genetic variation can drive the evolution of complex adaptive phenotypes. Positional cloning and candidate gene studies have identified a handful of regulatory and pigmentation genes implicated in Heliconius wing pattern variation, but little is known about the greater developmental networks within which these genes interact to pattern a wing. Here we took a large-scaletranscriptomic approach to identify the network of genes involved in Heliconius wing pattern development and variation. This included applying over 140 transcriptome microarrays to assay gene expression in dissected wing pattern elements across a range of developmental stages and wing pattern morphs of Heliconius erato. Results We identified a number of putative early prepattern genes with color-pattern related expression domains. We also identified 51 genes differentially expressed in association with natural color pattern variation. Of these, the previously identified color pattern “switch gene” optix was recovered as the first transcript to show color-specific differential expression. Most differentially expressed genes were transcribed late in pupal development and have roles in cuticle formation or pigment synthesis. These include previously undescribed transporter genes associated with ommochrome pigmentation. Furthermore, we observed upregulation of melanin-repressing genes such as ebony and Dat1 in non-melanic patterns. Conclusions This study identifies many new genes implicated in butterfly wing pattern development and provides a glimpse into the number and types of genes affected by variation in genes that drive color pattern evolution. PMID:22747837

The relationships between the levels of transcripts and the levels of the proteins they encode have not been examined comprehensively in mammals, although previous work in plants and yeast suggest a surprisingly modest correlation. We have examined this issue using a genetic approach in which natural variations were used to perturb both transcript levels and protein levels among inbred strains of mice. We quantified over 5,000 peptides and over 22,000 transcripts in livers of 97 inbred and recombinant inbred strains and focused on the 7,185 most heritable transcripts and 486 most reliable proteins. The transcript levels were quantified by microarray analysis in three replicates and the proteins were quantified by Liquid Chromatography–Mass Spectrometry using O(18)-reference-based isotope labeling approach. We show that the levels of transcripts and proteins correlate significantly for only about half of the genes tested, with an average correlation of 0.27, and the correlations of transcripts and proteins varied depending on the cellular location and biological function of the gene. We examined technical and biological factors that could contribute to the modest correlation. For example, differential splicing clearly affects the analyses for certain genes; but, based on deep sequencing, this does not substantially contribute to the overall estimate of the correlation. We also employed genome-wide association analyses to map loci controlling both transcript and protein levels. Surprisingly, little overlap was observed between the protein- and transcript-mapped loci. We have typed numerous clinically relevant traits among the strains, including adiposity, lipoprotein levels, and tissue parameters. Using correlation analysis, we found that a low number of clinical trait relationships are preserved between the protein and mRNA gene products and that the majority of such relationships are specific to either the protein levels or transcript levels. Surprisingly

Cynara cardunculus (2n = 2× = 34) is a member of the Asteraceae family that contributes significantly to the agricultural economy of the Mediterranean basin. The species includes two cultivated varieties, globe artichoke and cardoon, which are grown mainly for food. Cynara cardunculus is an orphan crop species whose genome/transcriptome has been relatively unexplored, especially in comparison to other Asteraceae crops. Hence, there is a significant need to improve its genomic resources through the identification of novel genes and sequence-based markers, to design new breeding schemes aimed at increasing quality and crop productivity. We report the outcome of cDNA sequencing and assembly for eleven accessions of C. cardunculus. Sequencing of three mapping parental genotypes using Roche 454-Titanium technology generated 1.7 × 10⁶ reads, which were assembled into 38,726 reference transcripts covering 32 Mbp. Putative enzyme-encoding genes were annotated using the KEGG-database. Transcription factors and candidate resistance genes were surveyed as well. Paired-end sequencing was done for cDNA libraries of eight other representative C. cardunculus accessions on an Illumina Genome Analyzer IIx, generating 46 × 10⁶ reads. Alignment of the IGA and 454 reads to reference transcripts led to the identification of 195,400 SNPs with a Bayesian probability exceeding 95%; a validation rate of 90% was obtained by Sanger-sequencing of a subset of contigs. These results demonstrate that the integration of data from different NGS platforms enables large-scaletranscriptome characterization, along with massive SNP discovery. This information will contribute to the dissection of key agricultural traits in C. cardunculus and facilitate the implementation of marker-assisted selection programs. PMID:22849342

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer. PMID:20126692

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer. PMID:20126692

We have developed an AFLP-based gene expression profiling method called ‘high coverage expression profiling’ (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to ∼4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs. Therefore, the method detects 70–80% of all transcripts, including non-coding transcripts, unknown and known genes. Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available. PMID:12907746

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. However, the genetic basis and regulatory mechanisms underlying the ability of S. oneidensis to survive and adapt to various environmentally relevant stresses is poorly understood. To define this organism's molecular response to elevated growth temperatures, temporal gene expression profiles were examined in cells subjected to heat stress using whole-genome DNA microarrays for S. oneidensis MR-1. Approximately 15 percent (711) of the predicted S. oneidensis genes represented on the microarray were significantly up- or down-regulated (P < 0.05) over a 25-min period following shift to the heat shock temperature (42 C). As expected, the majority of S. oneidensis genes exhibiting homology to known chaperones and heat shock proteins (Hsps) were highly and transiently induced. In addition, a number of predicted genes encoding enzymes in glycolys is and the pentose cycle, [NiFe] dehydrogenase, serine proteases, transcriptional regulators (MerR, LysR, and TetR families), histidine kinases, and hypothetical proteins were induced in response to heat stress. Genes encoding membrane proteins were differentially expressed, suggesting that cells possibly alter their membrane composition or structure in response to variations in growth temperature. A substantial number of the genes encoding ribosomal proteins displayed down-regulated co-expression patterns in response to heat stress, as did genes encoding prophage and flagellar proteins. Finally, based on computational comparative analysis of the upstream promoter regions of S.oneidensis heat-inducible genes, a putative regulatory motif, showing high conservation to the Escherichia coli sigma 32-binding consensus sequence, was identified.

The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. A time-series analysis of gene expression revealed changes in transcript levels of {approx}40% of genes ({approx}1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligosaccharide hydrolysis products

In this study, we undertook a functional characterization and transcriptomeanalysis that enabled a comprehensive study of the mating type loci of the mushroom Schizophyllum commune. Induced expression of both the bar2 receptor and the bap2(2) pheromone gene within 6 to 12 h after mates' contact was demonstrated by quantitative real-time PCR. Similar temporal expression patterns were confirmed for the allelic bbr1 receptor and bbp1 pheromone-encoding genes by Northern hybridization. Interestingly, the fusion of clamp connections to the subterminal cell was delayed in mating interactions in which one of the compatible partners expressed the bar2 receptor with a truncated C terminus. This developmental delay allowed the visualization of a green fluorescent protein (Gfp)-labeled truncated receptor at the cell periphery, consistent with a localization in the plasma membrane of unfused pseudoclamps. This finding does not support hypotheses envisioning a receptor localization to the nuclear membrane facilitating recognition between the two different nuclei present in each dikaryotic cell. Rather, Gfp fluorescence observed in such pseudoclamps indicated a role of receptor-pheromone interaction in clamp fusion. Transcriptome changes associated with mating interactions were analyzed in order to identify a role for pheromone-receptor interactions. We detected a total of 89 genes that were transcriptionally regulated in a mating type locus A-dependent manner, employing a cutoff of 5-fold changes in transcript abundance. Upregulation in cell cycle-related genes and downregulation of genes involved in metabolism were seen with this set of experiments. In contrast, mating type locus B-dependent transcriptome changes were observed in 208 genes, with a specific impact on genes related to cell wall and membrane metabolism, stress response, and the redox status of the cell. PMID:22210832

In the modern era of post genomics and transcriptomics, non-coding RNAs and non-coding regions of many RNAs are a big puzzle when we try deciphering their role in specific gene function. Gene function assessment is a main task wherein high throughput technologies provide an impressive body of data that enables the design of hypotheses linking genes to phenotypes. Gene knockdown technologies and RNA-dependent gene silencing are the most frequent approaches to assess the role of key effectors in a particular scenario. Ribozymes are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential. In our study, after an extensive transcriptomic search of Leishmania major transcriptome we found a Putative ATP dependent DNA helicase (Lmjf_09_0590) 3' UTR which has a structural signature similar to well-known HDV hammerhead ribozyme, even though they have variable sequence motifs. Henceforth, to determine their structural stability and sustainability we analyzed our predicted structural model of this 3'UTR with a 30ns MD simulation, further confirmed with 100ns MD simulation in presence of 5mM MgCl2 ionic environment. In this environment, structural stability was significantly improved by bonded interactions between a RNA backbone and Mg2+ ions. These predictions were further validated in silico using RNA normal mode analysis and anisotropic network modelling (ANM) studies. The study may be significantly imparted to know the functional importance of many such 3'UTRs to predict their role in a mechanistic manner. PMID:26901858

The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptomeanalysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases (CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction. PMID:24802510

Fused in sarcoma (FUS) is an RNA-binding protein that is causally associated with oncogenesis and neurodegeneration. Recently, the role of FUS in neurodegeneration has been extensively studied, because mutations in FUS are associated with amyotrophic lateral sclerosis (ALS), and the FUS protein has been identified as a major component of intracellular inclusions in neurodegenerative disorders including ALS and frontotemporal lobar degeneration. FUS is a key molecule in transcriptional regulation and RNA processing including processes such as pre-messenger RNA (mRNA) splicing and polyadenylation. Interaction of FUS with various components of the transcription machinery, spliceosome, and the 3'-end processing machinery has been identified. Furthermore, recent advances in high-throughput transcriptomic profiling approaches have enabled us to determine the mechanisms of FUS-dependent RNA processing networks at a cellular level. These analyses have revealed that depletion of FUS in neuronal cells affects alternative splicing and alternative polyadenylation of thousands of mRNAs. Gene ontology analysis has suggested that FUS-modulated genes are implicated in neuronal functions and development. CLIP-seq of FUS has shown that FUS is frequently clustered around these alternative sites of nascent RNA. ChIP-seq of RNA polymerase II (RNAP II) has demonstrated that an interaction between FUS and nascent RNA downregulates local transcriptional activity of RNAP II, which is critically involved in RNA processing. Both alternative splicing and alternative polyadenylation are fundamental processes by which cells expand their transcriptomic diversity, and are particularly essential in the nervous system. Dependence of transcriptomic diversity on FUS makes the nervous system vulnerable to neurodegeneration, when FUS is functionally compromised. WIREs RNA 2016, 7:330-340. doi: 10.1002/wrna.1338 For further resources related to this article, please visit the WIREs website. PMID:26822113

Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke. PMID:25375764

Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke. PMID:25375764

The sea cucumber Apostichopus japonicus Selenka 1867 represents an important resource in biomedical research, traditional medicine, and the seafood industry. Much of the commercial value of A. japonicus is determined by dorsal/ventral color variation (red, green, and black), yet the taxonomic relationships between these color variants are not clearly understood. We performed the first comparative analysis of de novo assembled transcriptome data from three color variants of A. japonicus. Using the Illumina platform, we sequenced nearly 177,596,774 clean reads representing a total of 18.2Gbp of sea cucumber transcriptome. A comparison of over 0.3 million transcript scaffolds against the Uniprot/Swiss-Prot database yielded 8513, 8602, and 8588 positive matches for green, red, and black body color transcriptomes, respectively. Using the Panther gene classification system, we assessed an extensive and diverse set of expressed genes in three color variants and found that (1) among the three color variants of A. japonicus, genes associated with RNA binding protein, oxidoreductase, nucleic acid binding, transferase, and KRAB box transcription factor were most commonly expressed; and (2) the main protein functional classes are differently regulated in all three color variants (extracellular matrix protein and phosphatase for green color, transporter and potassium channel for red color, and G-protein modulator and enzyme modulator for black color). This work will assist in the discovery and annotation of novel genes that play significant morphological and physiological roles in color variants of A. japonicus, and these sequence data will provide a useful set of resources for the rapidly growing sea cucumber aquaculture industry. PMID:27105969

Vibrio parahaemolyticus is the causative agent of food-borne gastroenteritis disease. Once consumed, human acid gastric fluid is perhaps one of the most important environmental stresses imposed on the bacterium. Herein, for the first time, we investigated Vibrio parahaemolyticus CHN25 response to artificial gastric fluid (AGF) stress by transcriptomicanalysis. The bacterium at logarithmic growth phase (LGP) displayed lower survival rates than that at stationary growth phase (SGP) under a sub-lethal acid condition (pH 4.9). Transcriptome data revealed that 11.6% of the expressed genes in Vibrio parahaemolyticus CHN25 was up-regulated in LGP cells after exposed to AGF (pH 4.9) for 30 min, including those involved in sugar transport, nitrogen metabolism, energy production and protein biosynthesis, whereas 14.0% of the genes was down-regulated, such as ATP-binding cassette (ABC) transporter and flagellar biosynthesis genes. In contrast, the AGF stress only elicited 3.4% of the genes from SGP cells, the majority of which were attenuated in expression. Moreover, the number of expressed regulator genes was also substantially reduced in SGP cells. Comparison of transcriptome profiles further revealed forty-one growth-phase independent genes in the AGF stress, however, half of which displayed distinct expression features between the two growth phases. Vibrio parahaemolyticus seemed to have evolved a number of molecular strategies for coping with the acid stress. The data here will facilitate future studies for environmental stresses and pathogenicity of the leading seafood-borne pathogen worldwide. PMID:25490137

The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptomeanalysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases (CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction. PMID:24802510

The response and adaption to salt remains poorly understood for beach morning glory [Ipomoea imperati (Vahl) Griseb], one of a few relatives of sweetpotato, known to thrive under salty and extreme drought conditions. In order to understand the genetic mechanisms underlying salt tolerance of a Convolvulaceae member, a genome-wide transcriptome study was carried out in beach morning glory by 454 pyrosequencing. A total of 286,584 filtered reads from both salt stressed and unstressed (control) root and shoot tissues were assembled into 95,790 unigenes with an average length of 667 base pairs (bp) and N50 of 706 bp. Putative differentially expressed genes (DEGs) were identified as transcripts overrepresented under salt stressed tissues compared to the control, and were placed into metabolic pathways. Most of these DEGs were involved in stress response, membrane transport, signal transduction, transcription activity and other cellular and molecular processes. We further analyzed the gene expression of 14 candidate genes of interest for salt tolerance through quantitative reverse transcription PCR (qRT-PCR) and confirmed their differential expression under salt stress in both beach morning glory and sweetpotato. The results comparing transcripts of I. imperati against the transcriptome of other Ipomoea species, including sweetpotato are also presented in this study. In addition, 6,233 SSR markers were identified, and an in silico analysis predicted that 434 primer pairs out of 4,897 target an identifiable homologous sequence in other Ipomoea transcriptomes, including sweetpotato. The data generated in this study will help in understanding the basics of salt tolerance of beach morning glory and the SSR resources generated will be useful for comparative genomics studies and further enhance the path to the marker-assisted breeding of sweetpotato for salt tolerance. PMID:26848754

Background Transcriptomeanalysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared ranki ngs for a priori identified physiological marker genes between the biofilm and published data sets. Results Biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. Transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. Similar rankings were obtained from data sets published in the GEO database http://www.ncbi.nlm.nih.gov/geo. By comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. These biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. Genes associated with elaboration of type IV pili were strongly expressed in the biofilm. The biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. Using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be 0.08 h-1. Conclusions Collectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity

Background Seaweeds of the Laurencia genus have a broad geographic distribution and are largely recognized as important sources of secondary metabolites, mainly halogenated compounds exhibiting diverse potential pharmacological activities and relevant ecological role as anti-epibiosis. Host-microbe interaction is a driving force for co-evolution in the marine environment, but molecular studies of seaweed-associated microbial communities are still rare. Despite the large amount of research describing the chemical compositions of Laurencia species, the genetic knowledge regarding this genus is currently restricted to taxonomic markers and general genome features. In this work we analyze the transcriptomic profile of L. dendroidea J. Agardh, unveil the genes involved on the biosynthesis of terpenoid compounds in this seaweed and explore the interactions between this host and its associated microbiome. Results A total of 6 transcriptomes were obtained from specimens of L. dendroidea sampled in three different coastal locations of the Rio de Janeiro state. Functional annotations revealed predominantly basic cellular metabolic pathways. Bacteria was the dominant active group in the microbiome of L. dendroidea, standing out nitrogen fixing Cyanobacteria and aerobic heterotrophic Proteobacteria. The analysis of the relative contribution of each domain highlighted bacterial features related to glycolysis, lipid and polysaccharide breakdown, and also recognition of seaweed surface and establishment of biofilm. Eukaryotic transcripts, on the other hand, were associated with photosynthesis, synthesis of carbohydrate reserves, and defense mechanisms, including the biosynthesis of terpenoids through the mevalonate-independent pathway. Conclusions This work describes the first transcriptomic profile of the red seaweed L. dendroidea, increasing the knowledge about ESTs from the Florideophyceae algal class. Our data suggest an important role for L. dendroidea in the primary

Background Pacific white shrimp (Litopenaeus vannamei), the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated. Methodology/Principal Findings This is the first study that used a next-generation high-throughput DNA sequencing technique, the Solexa/Illumina GA II method, to analyze the transcriptome from whole bodies of L. vannamei larvae. More than 2.4 Gb of raw data were generated, and 109,169 unigenes with a mean length of 396 bp were assembled using the SOAP denovo software. 73,505 unigenes (>200 bp) with good quality sequences were selected and subjected to annotation analysis, among which 37.80% can be matched in NCBI Nr database, 37.3% matched in Swissprot, and 44.1% matched in TrEMBL. Using BLAST and BLAST2Go softwares, 11,153 unigenes were classified into 25 Clusters of Orthologous Groups of proteins (COG) categories, 8171 unigenes were assigned into 51 Gene ontology (GO) functional groups, and 18,154 unigenes were divided into 220 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To primarily verify part of the results of assembly and annotations, 12 assembled unigenes that are homologous to many embryo development-related genes were chosen and subjected to RT-PCR for electrophoresis and Sanger sequencing analyses, and to real-time PCR for expression profile analyses during embryo development. Conclusions/Significance The L. vannamei transcriptome analyzed using the next-generation sequencing technique enriches the information of L. vannamei genes, which will facilitate our understanding of the genome background of crustaceans, and promote the studies on L. vannamei. PMID:23071809

Induced pluripotent stem cell (iPSC)-derived neurons and neural progenitors are great resources for studying neural development and differentiation and their disruptions in disease conditions, and hold the promise of future cell therapy. In general, iPSC lines can be established either specifically from patients with neuropsychiatric disorders or from healthy subjects. The iPSCs can then be induced to differentiate into neural lineages and the iPSC-derived neurons are valuable for various types of cell-based assays that seek to understand disease mechanisms and identify and test novel therapies. In addition, it is an ideal system for gene expression profiling (i.e., transcriptomicanalysis), an efficient and cost-effective way to explore the genetic programs regulating neurodevelopment. Moreover, transcriptomic comparison, which can be performed between patient-derived samples and controls, or in control lines in which the expression of specific genes has been disrupted, can uncover convergent gene targets and pathways that are downstream of the hundreds of candidate genes that have been associated with neuropsychiatric disorders. The results, especially after integration with spatiotemporal transcriptomic profiles of normal human brain development, have indeed helped to uncover gene networks, molecular pathways, and cellular signaling that likely play critical roles in disease development and progression. On the other hand, despite the great promise, many challenges remain in the usage of iPSC-derived neurons for modeling neuropsychiatric disorders, for example, how to generate relatively homogenous populations of specific neuronal subtypes that are affected in a particular disorder and how to better address the genetic heterogeneity that exists in the patient population. PMID:26631648

Background The antitumor drug daunorubicin exerts some of its cytotoxic effects by binding to DNA and inhibiting the transcription of different genes. We analysed this effect in vivo at the transcriptome level using the budding yeast Saccharomyces cerevisiae as a model and sublethal (IC40) concentrations of the drug to minimise general toxic effects. Results Daunorubicin affected a minor proportion (14%) of the yeast transcriptome, increasing the expression of 195 genes and reducing expression of 280 genes. Daunorubicin down-regulated genes included essentially all genes involved in the glycolytic pathway, the tricarboxylic acid cycle and alcohol metabolism, whereas transcription of ribosomal protein genes was not affected or even slightly increased. This pattern is consistent with a specific inhibition of glucose usage in treated cells, with only minor effects on proliferation or other basic cell functions. Analysis of promoters of down-regulated genes showed that they belong to a limited number of transcriptional regulatory units (regulons). Consistently, data mining showed that daunorubicin-induced changes in expression patterns were similar to those observed in yeast strains deleted for some transcription factors functionally related to the glycolysis and/or the cAMP regulatory pathway, which appeared to be particularly sensitive to daunorubicin. Conclusion The effects of daunorubicin treatment on the yeast transcriptome are consistent with a model in which this drug impairs binding of different transcription factors by competing for their DNA binding sequences, therefore limiting their effectiveness and affecting the corresponding regulatory networks. This proposed mechanism might have broad therapeutic implications against cancer cells growing under hypoxic conditions. PMID:18667070

In the modern era of post genomics and transcriptomics, non-coding RNAs and non-coding regions of many RNAs are a big puzzle when we try deciphering their role in specific gene function. Gene function assessment is a main task wherein high throughput technologies provide an impressive body of data that enables the design of hypotheses linking genes to phenotypes. Gene knockdown technologies and RNA-dependent gene silencing are the most frequent approaches to assess the role of key effectors in a particular scenario. Ribozymes are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential. In our study, after an extensive transcriptomic search of Leishmania major transcriptome we found a Putative ATP dependent DNA helicase (Lmjf_09_0590) 3’ UTR which has a structural signature similar to well-known HDV hammerhead ribozyme, even though they have variable sequence motifs. Henceforth, to determine their structural stability and sustainability we analyzed our predicted structural model of this 3’UTR with a 30ns MD simulation, further confirmed with 100ns MD simulation in presence of 5mM MgCl2 ionic environment. In this environment, structural stability was significantly improved by bonded interactions between a RNA backbone and Mg2+ ions. These predictions were further validated in silico using RNA normal mode analysis and anisotropic network modelling (ANM) studies. The study may be significantly imparted to know the functional importance of many such 3’UTRs to predict their role in a mechanistic manner. PMID:26901858

Syringa oblata Lindl. is a woody ornamental plant with high economic value and characteristics that include early flowering, multiple flower colors, and strong fragrance. Despite a long history of cultivation, the genetics and molecular biology of S. oblata are poorly understood. Transcriptome and expression profiling data are needed to identify genes and to better understand the biological mechanisms of floral pigments and scents in this species. Nine cDNA libraries were obtained from three replicates of three developmental stages: inflorescence with enlarged flower buds not protruded, inflorescence with corolla lobes not displayed, and inflorescence with flowers fully opened and emitting strong fragrance. Using the Illumina RNA-Seq technique, 319,425,972 clean reads were obtained and were assembled into 104,691 final unigenes (average length of 853 bp), 41.75% of which were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 36,967 were assigned to gene ontology categories and 19,956 were assigned to eukaryoticorthologous groups. Using the Kyoto Encyclopedia of Genes and Genomes pathway database, 12,388 unigenes were sorted into 286 pathways. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at different flower stages and that were related to floral pigment biosynthesis and fragrance metabolism. This comprehensive transcriptomicanalysis provides fundamental information on the genes and pathways involved in flower secondary metabolism and development in S. oblata, providing a useful database for further research on S. oblata and other plants of genus Syringa. PMID:26587670

Ophiocordyceps sinensis, also referred to as the Chinese caterpillar fungus, is a rare entomopathogenic fungus found in the Qinghai-Tibetan Plateau that is used as a traditional Chinese medicine. O. sinensis parasitizes the larvae of the ghost moth Thitarodes. Characterization of the transcriptome of O. sinensis before and after host infection may provide novel insight into the process by which the fungus interacts with Thitarodes and may help researchers understand how to sustain this valuable resource. In this study, we performed RNA-sequencing (RNA-seq) using Illumina HiSeqTM 2000 technology to generate gene expression profiles of two developmental stages of O. sinensis. Thread-like hyphae before infection and yeast-like hyphal bodies after infection of host larvae were collected for transcriptomeanalysis. We found that 1640 genes were differentially expressed (q-value analysis revealed that the differentially expressed genes (DEGs) were especially enriched in terms associated with Biological Process and Molecular Function. Several genes encoding transporter and permease proteins, three glycoside hydrolases, two mycotoxin-related proteins, an antigen protein, and an allergen were identified as being significantly up- or downregulated. Collectively, our findings provide a novel resource for understanding O. sinensis during two critical developmental stages, and offer the opportunity to further investigate the functional mechanisms underlying these stage-specific molecular differences. PMID:27268242

Caloric restriction (CR) has been shown to extend the lifespan of many species by improving cellular function and organismal health. Additionally, fat reduction by CR may play an important role in lengthening lifespan and preventing severe age-related diseases. Interestingly, CR induced the greatest transcriptome change in the epididymal fat of mice in our study. In this transcriptomeanalysis, we identified and categorized 446 genes that correlated with CR level. We observed down-regulation of several signaling pathways, including insulin/insulin-like growth factor 1 (insulin/IGF-1), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), and canonical wingless-type mouse mammary tumor virus integration site (Wnt). Many genes related to structural features, including extracellular matrix structure, cell adhesion, and the cytoskeleton, were down-regulated, with a strong correlation to the degree of CR. Furthermore, genes related to the cell cycle and adipogenesis were down-regulated. These biological processes are well-identified targets of insulin/IGF-1, EGF, TGF-β, and Wnt signaling. In contrast, genes involved in specific metabolic processes, including the tricarboxylic acid cycle and the electron transport chain were up-regulated. We performed in silico analysis of the promoter sequences of CR-responsive genes and identified two associated transcription factors, Paired-like homeodomain 2 (Pitx2) and Paired box gene 6 (Pax6). Our results suggest that strict regulation of signaling pathways is critical for creating the optimal energy homeostasis to extend lifespan. PMID:26606930

Drought and salinity are the major environmental factors that affect rice productivity. Comparative transcriptomeanalysis between tolerant and sensitive rice cultivars can provide insights into the regulatory mechanisms involved in these stress responses. In this study, the comparison of transcriptomes of a drought-tolerant [Nagina 22 (N22)] and a salinity-tolerant (Pokkali) rice cultivar with IR64 (susceptible cultivar) revealed variable transcriptional responses under control and stress conditions. A total of 801 and 507 transcripts were exclusively differentially expressed in N22 and Pokkali rice cultivars, respectively, under stress conditions. Gene ontology analysis suggested the enrichment of transcripts involved in response to abiotic stress and regulation of gene expression in stress-tolerant rice cultivars. A larger number of transcripts encoding for members of NAC and DBP transcription factor (TF) families in N22 and members of bHLH and C2H2 TF families in Pokkali exhibited differential regulation under desiccation and salinity stresses, respectively. Transcripts encoding for thioredoxin and involved in phenylpropanoid metabolism were up-regulated in N22, whereas transcripts involved in wax and terpenoid metabolism were up-regulated in Pokkali. Overall, common and cultivar-specific stress-responsive transcripts identified in this study can serve as a helpful resource to explore novel candidate genes for abiotic stress tolerance in rice. PMID:27029818

Radix polygalae, the dried roots of Polygala tenuifolia and P. sibirica, is one of the most well-known traditional Chinese medicinal plants. Radix polygalae contains various saponins, xanthones, and oligosaccharide esters and these compounds are responsible for several pharmacological properties. To provide basic breeding information, enhance molecular biological analysis, and determine secondary metabolite biosynthetic pathways of P. tenuifolia, we applied Illumina sequencing technology and de novo assembly. We also applied this technique to gain an overview of P. tenuifolia transcriptome from samples with different years. Using Illumina sequencing, approximately 67.2% of unique sequences were annotated by basic local alignment search tool similarity searches against public sequence databases. We classified the annotated unigenes by using Nr, Nt, GO, COG, and KEGG databases compared with NCBI. We also obtained many candidates CYP450s and UGTs by the analysis of genes in the secondary metabolite biosynthetic pathways, including putative terpenoid backbone and phenylpropanoid biosynthesis pathway. With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of P. tenuifolia may be improved. Genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of P. tenuifolia in pharmaceutical industries. PMID:26543847

Radix polygalae, the dried roots of Polygala tenuifolia and P. sibirica, is one of the most well-known traditional Chinese medicinal plants. Radix polygalae contains various saponins, xanthones, and oligosaccharide esters and these compounds are responsible for several pharmacological properties. To provide basic breeding information, enhance molecular biological analysis, and determine secondary metabolite biosynthetic pathways of P. tenuifolia, we applied Illumina sequencing technology and de novo assembly. We also applied this technique to gain an overview of P. tenuifolia transcriptome from samples with different years. Using Illumina sequencing, approximately 67.2% of unique sequences were annotated by basic local alignment search tool similarity searches against public sequence databases. We classified the annotated unigenes by using Nr, Nt, GO, COG, and KEGG databases compared with NCBI. We also obtained many candidates CYP450s and UGTs by the analysis of genes in the secondary metabolite biosynthetic pathways, including putative terpenoid backbone and phenylpropanoid biosynthesis pathway. With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of P. tenuifolia may be improved. Genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of P. tenuifolia in pharmaceutical industries. PMID:26543847

Helminth infections affect more than a third of the world’s population. Despite very broad phylogenetic differences among helminth parasite species, a systemic Th2 host immune response is typically associated with long-term helminth infections, also known as the “helminth effect”. Many investigations have been carried out to study host gene expression profiles during helminth infections. The objective of this study is to determine if there is a common transcriptomic signature characteristic of the helminth effect across multiple helminth species and tissue types. To this end, we performed a comprehensive meta-analysis of publicly available gene expression datasets. After data processing and adjusting for study-specific effects, we identified ~700 differentially expressed genes that are changed consistently during helminth infections. Functional enrichment analyses indicate that upregulated genes are predominantly involved in various immune functions, including immunomodulation, immune signaling, inflammation, pathogen recognition and antigen presentation. Down-regulated genes are mainly involved in metabolic process, with only a few of them are involved in immune regulation. This common immune gene signature confirms previous observations and indicates that the helminth effect is robust across different parasite species as well as host tissue types. To the best of our knowledge, this study is the first comprehensive meta-analysis of host transcriptome profiles during helminth infections. PMID:27058578

Bryophytes, the most basal of the extant land plants, diverged at least 450 million years ago. A major feature of these plants is the biphasic alternation of generations between a dominant haploid gametophyte and a minor diploid sporophyte phase. These dramatic differences in form and function occur in a constant genetic background, raising the question of whether the switch from gametophyte-to-sporophyte development reflects major changes in the spectrum of genes being expressed or alternatively whether only limited changes in gene expression occur and the differences in plant form are due to differences in how the gene products are put together. This study performed replicated microarray analyses of RNA from several thousand dissected and developmentally staged sporophytes of the moss Physcomitrella patens, allowing analysis of the transcriptomes of the sporophyte and early gametophyte, as well as the early stages of moss sporophyte development. The data indicate that more significant changes in transcript profile occur during the switch from gametophyte to sporophyte than recently reported, with over 12% of the entire transcriptome of P. patens being altered during this major developmental transition. Analysis of the types of genes contributing to these differences supports the view of the early sporophyte being energetically and nutritionally dependent on the gametophyte, provides a profile of homologues to genes involved in angiosperm stomatal development and physiology which suggests a deeply conserved mechanism of stomatal control, and identifies a novel series of transcription factors associated with moss sporophyte development. PMID:23888066

Eggplant is the third most important Solanaceae crop after tomato and potato, particularly in India and China. A transcriptomeanalysis of eggplant's fruit was performed to study genes involved in medicinal importance and allergies. Illumina HiSeq 2000 system generated 89,763,638 raw reads (~18 Gb) from eggplant. High quality reads (59,039,694) obtained after trimming process, were assembled into a total of 149,224 non redundant set of transcripts. Out of 80,482 annotated sequences of eggplant fruit (BLASTx results against nr-green plant database), 40,752 transcripts showed significant similarity with predicted proteins of Solanum tuberosum (51%) followed by Solanum lycopersicum (34%) and other sequenced plant genomes. With BLASTx top hit analysis against existing allergens, a total of 1986 homologous allergen sequences were found, which had >37% similarity with 48 different allergens existing in the database. From the 48 putative allergens, 526 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. Transcript sequences generated from this study can be used to map epitopes of monoclonal antibodies and polyclonal sera from patients. With the support of this whole transcriptome catalogue of eggplant fruit, complete list of genes can be predicted based on which secondary structures of proteins may be modeled. PMID:26424595

A large number of plants have been tested and exploited in search of a green chemistry approach for the fabrication of gold or other precious metal nanomaterials. Despite the potential of plant based methods, very little is known about the underlying biochemical reactions and genes involved in the biotransformation mechanism of AuCl4 into gold nanoparticles (AuNPs). In this research, we thus focused on studying the effect of Au on growth and nanoparticles formation by analyses of transcriptome, proteome and ionome shift in Arabidopsis. Au exposure favored the growth of Arabidopsis seedling and induced formation of nanoparticles in root and shoot, as indicated by optical and hyperspectral imaging. Root transcriptomeanalysis demonstrated the differential expression of the members of WRKY, MYB and BHLH gene families, which are involved in the Fe and other essential metals homeostasis. The proteome analysis revealed that Glutathione S-transferases were induced in the shoot and suggested its potential role in the biosynthesis AuNPs. This study also demonstrated the role of plant hormone auxin in determining the Au induced root system architecture. This is the first study using an integrated approach to understand the in planta biotransformation of KAuCl4 into AuNPs. PMID:26902325

Phycocyanin (PC) from Spirulina platensis has inhibitory effects on tumor cell growth. In this research, the transcriptome study was designed to investigate the underlying molecular mechanisms of PC inhibition on human ovarian cancer cell SKOV-3 proliferation. The PC IC50 was 216.6μM and 163.8μM for 24h and 48h exposure, respectively, as determined by CCK-8 assay. The morphological changes of SKOV-3 cells after PC exposure were recorded using HE staining. Cells arrested in G2/M stages as determined by flow cytometry. The transcriptomeanalysis showed that 2031 genes (with > three-fold differences) were differentially expressed between the untreated and the PC-treated cells, including 1065 up-regulated and 966 down-regulated genes. Gene ontology and KEGG pathway analysis identified 18 classical pathways that were remarkably enriched, such as neurotrophin signaling pathway, VEGF signaling pathway and P53 signaling pathway. qPCR results further showed that PTPN12, S100A2, RPL26, and LAMA3 increased while HNRNPA1P10 decreased in PC-treated cells. Molecules and genes in those pathways may be potential targets to develop treatments for ovarian cancer. PMID:26995654

Transcriptome dynamics in the longissimus muscle (LM) of young Angus cattle were evaluated at 0, 60, 120, and 220 days from early-weaning. Bioinformatic analysis was performed using the dynamic impact approach (DIA) by means of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Database for Annotation, Visualization and Integrated Discovery (DAVID) databases. Between 0 to 120 days (growing phase) most of the highly-impacted pathways (eg, ascorbate and aldarate metabolism, drug metabolism, cytochrome P450 and Retinol metabolism) were inhibited. The phase between 120 to 220 days (finishing phase) was characterized by the most striking differences with 3,784 differentially expressed genes (DEGs). Analysis of those DEGs revealed that the most impacted KEGG canonical pathway was glycosylphosphatidylinositol (GPI)-anchor biosynthesis, which was inhibited. Furthermore, inhibition of calpastatin and activation of tyrosine aminotransferase ubiquitination at 220 days promotes proteasomal degradation, while the concurrent activation of ribosomal proteins promotes protein synthesis. Therefore, the balance of these processes likely results in a steady-state of protein turnover during the finishing phase. Results underscore the importance of transcriptome dynamics in LM during growth. PMID:23943656

Marine sandy beaches represent dynamic environments often subject to harsh conditions and climate fluctuations, where natural and anthropogenic inputs of freshwater from fluvial and pluvial sources alter salinity, which has been recognized as a key variable affecting the distribution of aquatic organisms and influencing critical physiological processes. The marine arenicolous fungus Corollospora maritima is a worldwide-distributed saprobe that has been reported to present tolerance to freshwater. Here, we present a transcriptomeanalysis that will provide the first insight of the genomic content for this fungus and a gene expression comparison between two different salinity conditions. We also identified genes that are candidates for being differentially expressed in response to environmental variations on salinity during the fungal growth. The de novo reconstruction of C. maritima transcriptome Illumina sequencing provided a total of 14,530 transcripts (16 megabases). The comparison between the two growth conditions rendered 103 genes specifically overexpressed in seawater, and 132 genes specifically up-regulated under freshwater. Using fungal isolates collected from different beaches, the specific environmental regulation of particular transcript differential expression was confirmed by RT-qPCR. To our knowledge, this is the first analysis that explores the marine fungus C. maritima molecular responses to overcome freshwater stress, and these data could shed light to understand the fungal adaptation and plasticity mechanisms to the marine habitat. PMID:26116293

Rhizome is the storage organ of lotus derived from modified stems. The development of rhizome is a complex process and depends on the balanced expression of the genes that is controlled by environmental and endogenous factors. However, little is known about the mechanism that regulates rhizome girth enlargement. In this study, using RNA-seq, transcriptomic analyses were performed at three rhizome developmental stages-the stolon, middle swelling and later swelling stage -in the cultivars 'ZO' (temperate lotus with enlarged rhizome) and 'RL' (tropical lotus with stolon). About 348 million high-quality reads were generated, and 88.5% of the data were mapped to the reference genome. Of 26783 genes identified, 24069 genes were previously predicted in the reference, and 2714 genes were novel transcripts. Moreover, 8821 genes were differentially expressed between the cultivars at the three stages. Functional analysis identified that these genes were significantly enriched in pathways carbohydrate metabolism and plant hormone signal transduction. Twenty-two genes involved in photoperiod pathway, starch metabolism and hormone signal transduction were candidate genes inducing rhizome girth enlargement. Comparative transcriptomicanalysis detected several differentially expressed genes and potential candidate genes required for rhizome girth enlargement, which lay a foundation for future studies on molecular mechanisms underlying rhizome formation. PMID:26279185

Rhizome is the storage organ of lotus derived from modified stems. The development of rhizome is a complex process and depends on the balanced expression of the genes that is controlled by environmental and endogenous factors. However, little is known about the mechanism that regulates rhizome girth enlargement. In this study, using RNA-seq, transcriptomic analyses were performed at three rhizome developmental stages—the stolon, middle swelling and later swelling stage —in the cultivars ‘ZO’ (temperate lotus with enlarged rhizome) and ‘RL’ (tropical lotus with stolon). About 348 million high-quality reads were generated, and 88.5% of the data were mapped to the reference genome. Of 26783 genes identified, 24069 genes were previously predicted in the reference, and 2714 genes were novel transcripts. Moreover, 8821 genes were differentially expressed between the cultivars at the three stages. Functional analysis identified that these genes were significantly enriched in pathways carbohydrate metabolism and plant hormone signal transduction. Twenty-two genes involved in photoperiod pathway, starch metabolism and hormone signal transduction were candidate genes inducing rhizome girth enlargement. Comparative transcriptomicanalysis detected several differentially expressed genes and potential candidate genes required for rhizome girth enlargement, which lay a foundation for future studies on molecular mechanisms underlying rhizome formation. PMID:26279185

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models. PMID:26967054

Drought and salinity are the major environmental factors that affect rice productivity. Comparative transcriptomeanalysis between tolerant and sensitive rice cultivars can provide insights into the regulatory mechanisms involved in these stress responses. In this study, the comparison of transcriptomes of a drought-tolerant [Nagina 22 (N22)] and a salinity-tolerant (Pokkali) rice cultivar with IR64 (susceptible cultivar) revealed variable transcriptional responses under control and stress conditions. A total of 801 and 507 transcripts were exclusively differentially expressed in N22 and Pokkali rice cultivars, respectively, under stress conditions. Gene ontology analysis suggested the enrichment of transcripts involved in response to abiotic stress and regulation of gene expression in stress-tolerant rice cultivars. A larger number of transcripts encoding for members of NAC and DBP transcription factor (TF) families in N22 and members of bHLH and C2H2 TF families in Pokkali exhibited differential regulation under desiccation and salinity stresses, respectively. Transcripts encoding for thioredoxin and involved in phenylpropanoid metabolism were up-regulated in N22, whereas transcripts involved in wax and terpenoid metabolism were up-regulated in Pokkali. Overall, common and cultivar-specific stress-responsive transcripts identified in this study can serve as a helpful resource to explore novel candidate genes for abiotic stress tolerance in rice. PMID:27029818

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models. PMID:26967054

The neonatal management of preterm born infants often results in damage to the developing lung and subsequent morbidity, referred to as bronchopulmonary dysplasia (BPD). Animal models may help in understanding the molecular processes involved in this condition and define therapeutic targets. Our goal was to identify molecular pathways using the earlier described preterm rabbit model of hyperoxia induced lung-injury. Transcriptomeanalysis by mRNA-sequencing was performed on lungs from preterm rabbit pups born at day 28 of gestation (term: 31 days) and kept in hyperoxia (95% O2) for 7 days. Controls were preterm pups kept in normoxia. Transcriptomic data were analyzed using Array Studio and Ingenuity Pathway Analysis (IPA), in order to identify the central molecules responsible for the observed transcriptional changes. We detected 2217 significantly dysregulated transcripts following hyperoxia, of which 90% could be identified. Major pathophysiological dysregulations were found in inflammation, lung development, vascular development and reactive oxygen species (ROS) metabolism. To conclude, amongst the many dysregulated transcripts, major changes were found in the inflammatory, oxidative stress and lung developmental pathways. This information may be used for the generation of new treatment hypotheses for hyperoxia-induced lung injury and BPD. PMID:26317699

Pseudostellariae Radix is an important traditional Chinese medicine (TCM), which is consumed commonly for its positive health effects. However, a lack of transcriptomic and genomic information hinders research on Pseudostellariae Radix. Here, high-throughput RNA sequencing (RNA-seq) was employed for the de novo assembly to analyze the transcriptome in Pseudostellariae Radix, finding significantly differentially expressed genes in this TCM from different fields based on RNA-seq and bioinformatic analysis. A total of 146,408,539 paired-end reads were generated and assembled into 89,857 unigenes with an average length of 862bp. All of the assembly unigenes were annotated by running BLASTx and BLASTn similarity searches on the Non-redundant nucleotide database (NT), the Non-redundant protein database (NR), Swiss-Prot, Cluster of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Interpro. On the basis of bioinformatic analysis and the expression profiles for Pseudostellariae Radix, 29 significantly differentially expressed genes were identified, which provides the basic information for exploring the molecular mechanisms that determine the quality of Pseudostellariae Radix from different fields. The expression levels of 29 genes were validated by real-time quantitative PCR (RT-qPCR). This is the first study to sample Pseudostellariae Radix, which provides an invaluable resource for understanding the genome of this herb. PMID:27125225

Fathead minnow and zebrafish are among the most intensively studied fish species in environmental toxicogenomics. To aid the assessment and interpretation of subtle transcriptomic effects from treatment conditions of interest, better characterization and understanding are needed for natural variation in gene expression among fish individuals from lab cultures. Leveraging the transcriptomics data from a number of our toxicogenomics studies conducted over the years, we conducted a meta-analysis of nearly 600 microarrays generated from the ovary tissue of untreated, reproductively mature fathead minnow and zebrafish samples. As expected, there was considerable batch-to-batch transcriptomic variation; this “batch-effect” appeared to differentially impact subsets of fish transcriptomes in a nonsystematic way. Temporally more closely spaced batches tended to share a greater transcriptomic similarity among one another. The overall level of within-batch variation was quite low in fish ovary tissue, making it a suitable system for studying chemical stressors with subtle biological effects. The observed differences in the within-batch variability of gene expression, at the levels of both individual genes and pathways, were probably both technical and biological. This suggests that biological interpretation and prioritization of genes and pathways targeted by experimental conditions should take into account both their intrinsic variability and the size of induced transcriptional changes. There was significant conservation of both the genomes and transcriptomes between fathead minnow and zebrafish. The high degree of conservation offers promising opportunities in not only studying fish molecular responses to environmental stressors by a comparative biology approach, but also effective sharing of a large amount of existing public transcriptomics data for developing toxicogenomics applications. PMID:25493933

The biological function of the Prion protein remains largely unknown but recent data revealed its implication in early zebrafish and mammalian embryogenesis. To gain further insight into its biological function, comparative transcriptomicanalysis between FVB/N and FVB/N Prnp knockout mice was performed at early embryonic stages. RNAseq analysis revealed the differential expression of 73 and 263 genes at E6.5 and E7.5, respectively. The related metabolic pathways identified in this analysis partially overlap with those described in PrP1 and PrP2 knockdown zebrafish embryos and prion-infected mammalian brains and emphasize a potentially important role for the PrP family genes in early developmental processes. PMID:21858045

Background Body color and coloration patterns are important phenotypic traits to maintain survival and reproduction activities. The Oujiang color varieties of common carp (Cyprinus carpio var. color), with a narrow distribution in Zhejiang Province of China and a history of aquaculture for over 1,200 years, consistently exhibit a variety of body color patterns. The molecular mechanism underlying diverse color patterns in these variants is unknown. To the practical end, it is essential to develop molecular markers that can distinguish different phenotypes and assist selective breeding. Methodology/Principal Findings In this exploratory study, we conducted Roche 454 transcriptome sequencing of two pooled skin tissue samples of Oujiang common carp, which correspond to distinct color patterns, red with big black spots (RB) and whole white (WW), and a total of 737,525 sequence reads were generated. The reads obtained in this study were co-assembled jointly with common carp Roche 454 sequencing reads downloaded from NCBI SRA database, resulting in 43,923 isotigs and 546,676 singletons. Over 31 thousand (31,445; 71.6%) isotigs were found with significant BLAST matches (E<1e-10) to the nr protein database, which corresponds to 12,597 annotated zebrafish genes. A total of 70,947 isotigs and singletons (transcripts) were annotated with Gene Ontology, and 60,221 transcripts were found with corresponding EC numbers. Out of 145 zebrafish pigmentation genes, orthologs for 117 were recovered in Oujiang color carp transcriptome, including 18 found only among singletons. Our transcriptomeanalysis revealed over 52,902 SNPs in Oujiang common carp, and identified 63 SNP markers that are putatively unique either for RB or WW. Conclusions The transcriptome of Oujiang color varieties of common carp obtained through this study, along with the pigmentation genes recovered and the color pattern-specific molecular markers developed, will facilitate future research on the molecular mechanism

Oryza meyeriana (O. meyeriana), with a GG genome type (2n = 24), accumulated plentiful excellent characteristics with respect to resistance to many diseases such as rice shade and blast, even immunity to bacterial blight. It is very important to know if the diseases-resistant genes exist and express in this wild rice under native conditions. However, limited genomic or transcriptomic data of O. meyeriana are currently available. In this study, we present the first comprehensive characterization of the O. meyeriana transcriptome using RNA-seq and obtained 185,323 contigs with an average length of 1,692 bp and an N50 of 2,391 bp. Through differential expression analysis, it was found that there were most tissue-specifically expressed genes in roots, and next to stems and leaves. By similarity search against protein databases, 146,450 had at least a significant alignment to existed gene models. Comparison with the Oryza sativa (japonica-type Nipponbare and indica-type 93–11) genomes revealed that 13% of the O. meyeriana contigs had not been detected in O. sativa. Many diseases-resistant genes, such as bacterial blight resistant, blast resistant, rust resistant, fusarium resistant, cyst nematode resistant and downy mildew gene, were mined from the transcriptomic database. There are two kinds of rice bacterial blight-resistant genes (Xa1 and Xa26) differentially or specifically expressed in O. meyeriana. The 4 Xa1 contigs were all only expressed in root, while three of Xa26 contigs have the highest expression level in leaves, two of Xa26 contigs have the highest expression profile in stems and one of Xa26 contigs was expressed dominantly in roots. The transcriptomic database of O. meyeriana has been constructed and many diseases-resistant genes were found to express under native condition, which provides a foundation for future discovery of a number of novel genes and provides a basis for studying the molecular mechanisms associated with disease resistance in O

Background Teladorsagia circumcincta (order Strongylida) is an economically important parasitic nematode of small ruminants (including sheep and goats) in temperate climatic regions of the world. Improved insights into the molecular biology of this parasite could underpin alternative methods required to control this and related parasites, in order to circumvent major problems associated with anthelmintic resistance. The aims of the present study were to define the transcriptome of the adult stage of T. circumcincta and to infer the main pathways linked to molecules known to be expressed in this nematode. Since sheep develop acquired immunity against T. circumcincta, there is some potential for the development of a vaccine against this parasite. Hence, we infer excretory/secretory molecules for T. circumcincta as possible immunogens and vaccine candidates. Results A total of 407,357 ESTs were assembled yielding 39,852 putative gene sequences. Conceptual translation predicted 24,013 proteins, which were then subjected to detailed annotation which included pathway mapping of predicted proteins (including 112 excreted/secreted [ES] and 226 transmembrane peptides), domain analysis and GO annotation was carried out using InterProScan along with BLAST2GO. Further analysis was carried out for secretory signal peptides using SignalP and non-classical sec pathway using SecretomeP tools. For ES proteins, key pathways, including Fc epsilon RI, T cell receptor, and chemokine signalling as well as leukocyte transendothelial migration were inferred to be linked to immune responses, along with other pathways related to neurodegenerative diseases and infectious diseases, which warrant detailed future studies. KAAS could identify new and updated pathways like phagosome and protein processing in endoplasmic reticulum. Domain analysis for the assembled dataset revealed families of serine, cysteine and proteinase inhibitors which might represent targets for parasite intervention. Inter

Polymyxin B and colistin are exclusively active against Gram-negative pathogens and have been used in the clinic as a last-line therapy. In this study, we investigated the antimicrobial activity of a novel polymyxin, FADDI-019, against Staphylococcus aureus. MIC and time-kill assays were employed to measure the activity of FADDI-019 against S. aureus ATCC 700699. Cell morphology was examined with scanning electron microscopy (SEM), and cell membrane polarity was measured using flow cytometry. Transcriptome changes caused by FADDI-019 treatment were investigated using transcriptome sequencing (RNA-Seq). Pathway analysis was conducted to examine the mechanism of the antibacterial activity of FADDI-019 and to rationally design a synergistic combination. Polymyxin B and colistin were not active against S. aureus strains with MICs of >128 mg/liter; however, FADDI-019 had a MIC of 16 mg/liter. Time-kill assays revealed that no S. aureus regrowth was observed after 24 h at 2× to 4× MIC of FADDI-019. Scanning electron microscopy (SEM) and flow cytometry results indicated that FADDI-019 treatment had no effect on cell morphology but caused membrane depolarization. The vancomycin resistance genes vraRS, as well as the VraRS regulon, were activated by FADDI-019. Virulence determinants controlled by SaeRS and the expression of enterotoxin genes yent2, sei, sem, and seo were significantly downregulated by FADDI-019. Pathway analysis of transcriptomic data was predictive of a synergistic combination comprising FADDI-019 and sulfamethoxazole. Our study is the first to examine the mechanism of the killing of a novel polymyxin against S. aureus. We also show the potential of transcriptomic and pathway analysis as tools to design synergistic antibiotic combinations. IMPORTANCE S. aureus is currently one of the most pervasive multidrug-resistant pathogens and commonly causes nosocomial infections. Clinicians are faced with a dwindling armamentarium to treat infections

ABSTRACT Polymyxin B and colistin are exclusively active against Gram-negative pathogens and have been used in the clinic as a last-line therapy. In this study, we investigated the antimicrobial activity of a novel polymyxin, FADDI-019, against Staphylococcus aureus. MIC and time-kill assays were employed to measure the activity of FADDI-019 against S. aureus ATCC 700699. Cell morphology was examined with scanning electron microscopy (SEM), and cell membrane polarity was measured using flow cytometry. Transcriptome changes caused by FADDI-019 treatment were investigated using transcriptome sequencing (RNA-Seq). Pathway analysis was conducted to examine the mechanism of the antibacterial activity of FADDI-019 and to rationally design a synergistic combination. Polymyxin B and colistin were not active against S. aureus strains with MICs of >128 mg/liter; however, FADDI-019 had a MIC of 16 mg/liter. Time-kill assays revealed that no S. aureus regrowth was observed after 24 h at 2× to 4× MIC of FADDI-019. Scanning electron microscopy (SEM) and flow cytometry results indicated that FADDI-019 treatment had no effect on cell morphology but caused membrane depolarization. The vancomycin resistance genes vraRS, as well as the VraRS regulon, were activated by FADDI-019. Virulence determinants controlled by SaeRS and the expression of enterotoxin genes yent2, sei, sem, and seo were significantly downregulated by FADDI-019. Pathway analysis of transcriptomic data was predictive of a synergistic combination comprising FADDI-019 and sulfamethoxazole. Our study is the first to examine the mechanism of the killing of a novel polymyxin against S. aureus. We also show the potential of transcriptomic and pathway analysis as tools to design synergistic antibiotic combinations. IMPORTANCE S. aureus is currently one of the most pervasive multidrug-resistant pathogens and commonly causes nosocomial infections. Clinicians are faced with a dwindling armamentarium to treat

Studies using the placental transcriptome to identify key molecules relevant for preeclampsia are hampered by a relatively small sample size. In addition, they use a variety of bioinformatics and statistical methods, making comparison of findings challenging. To generate a more robust preeclampsia gene expression signature, we performed a meta-analysis on the original data of 11 placenta RNA microarray experiments, representing 139 normotensive and 116 preeclamptic pregnancies. Microarray data were pre-processed and analyzed using standardized bioinformatics and statistical procedures and the effect sizes were combined using an inverse-variance random-effects model. Interactions between genes in the resulting gene expression signature were identified by pathway analysis (Ingenuity Pathway Analysis, Gene Set Enrichment Analysis, Graphite) and protein-protein associations (STRING). This approach has resulted in a comprehensive list of differentially expressed genes that led to a 388-gene meta-signature of preeclamptic placenta. Pathway analysis highlights the involvement of the previously identified hypoxia/HIF1A pathway in the establishment of the preeclamptic gene expression profile, while analysis of protein interaction networks indicates CREBBP/EP300 as a novel element central to the preeclamptic placental transcriptome. In addition, there is an apparent high incidence of preeclampsia in women carrying a child with a mutation in CREBBP/EP300 (Rubinstein-Taybi Syndrome). The 388-gene preeclampsia meta-signature offers a vital starting point for further studies into the relevance of these genes (in particular CREBBP/EP300) and their concomitant pathways as biomarkers or functional molecules in preeclampsia. This will result in a better understanding of the molecular basis of this disease and opens up the opportunity to develop rational therapies targeting the placental dysfunction causal to preeclampsia. PMID:26171964

Studies using the placental transcriptome to identify key molecules relevant for preeclampsia are hampered by a relatively small sample size. In addition, they use a variety of bioinformatics and statistical methods, making comparison of findings challenging. To generate a more robust preeclampsia gene expression signature, we performed a meta-analysis on the original data of 11 placenta RNA microarray experiments, representing 139 normotensive and 116 preeclamptic pregnancies. Microarray data were pre-processed and analyzed using standardized bioinformatics and statistical procedures and the effect sizes were combined using an inverse-variance random-effects model. Interactions between genes in the resulting gene expression signature were identified by pathway analysis (Ingenuity Pathway Analysis, Gene Set Enrichment Analysis, Graphite) and protein-protein associations (STRING). This approach has resulted in a comprehensive list of differentially expressed genes that led to a 388-gene meta-signature of preeclamptic placenta. Pathway analysis highlights the involvement of the previously identified hypoxia/HIF1A pathway in the establishment of the preeclamptic gene expression profile, while analysis of protein interaction networks indicates CREBBP/EP300 as a novel element central to the preeclamptic placental transcriptome. In addition, there is an apparent high incidence of preeclampsia in women carrying a child with a mutation in CREBBP/EP300 (Rubinstein-Taybi Syndrome). The 388-gene preeclampsia meta-signature offers a vital starting point for further studies into the relevance of these genes (in particular CREBBP/EP300) and their concomitant pathways as biomarkers or functional molecules in preeclampsia. This will result in a better understanding of the molecular basis of this disease and opens up the opportunity to develop rational therapies targeting the placental dysfunction causal to preeclampsia. PMID:26171964

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptomeanalysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptomeanalysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence. Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin," in which we successfully performed the first complete transcriptomeanalysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases. The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with

Leaves are vital organs for biomass and seed production because of their role in the generation of metabolic energy and organic compounds. A better understanding of the molecular networks underlying leaf development is crucial to sustain global requirements for food and renewable energy. Here, we combined transcriptome profiling of proliferative leaf tissue with in-depth phenotyping of the fourth leaf at later stages of development in 197 recombinant inbred lines of two different maize (Zea mays) populations. Previously, correlation analysis in a classical biparental mapping population identified 1,740 genes correlated with at least one of 14 traits. Here, we extended these results with data from a multiparent advanced generation intercross population. As expected, the phenotypic variability was found to be larger in the latter population than in the biparental population, although general conclusions on the correlations among the traits are comparable. Data integration from the two diverse populations allowed us to identify a set of 226 genes that are robustly associated with diverse leaf traits. This set of genes is enriched for transcriptional regulators and genes involved in protein synthesis and cell wall metabolism. In order to investigate the molecular network context of the candidate gene set, we integrated our data with publicly available functional genomics data and identified a growth regulatory network of 185 genes. Our results illustrate the power of combining in-depth phenotyping with transcriptomics in mapping populations to dissect the genetic control of complex traits and present a set of candidate genes for use in biomass improvement. PMID:26754667

The detailed molecular mechanism of Bmovo-1 regulation of ovary size is unclear. To uncover the mechanism of Bmovo-1 regulation of ovarian development and oogenesis using RNA-Seq, we compared the transcriptomes of wild type (WT) and Bmovo-1-overexpressing silkworm (silkworm+Bmovo-1) ovaries. Using a pair-end Illumina Solexa sequencing strategy, 5,296,942 total reads were obtained from silkworm+Bmovo-1 ovaries and 6,306,078 from WT ovaries. The average read length was about 100 bp. Clean read ratios were 98.79% for silkworm+Bmovo-1 and 98.87% for WT silkworm ovaries. Comparative transcriptomeanalysis showed 123 upregulated and 111 downregulated genes in silkworm+Bmovo-1 ovaries. These differentially expressed genes were enriched in the extracellular and extracellular spaces and involved in metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems. Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development and oogenesis and oocyte proliferation and transport of nutrients to ovaries by altering nutrient partitioning, which would support ovary development. Excessive consumption of nutrients for ovary development alters nutrient partitioning and deters silk protein synthesis. PMID:26643037

Osmotic stress is one of the most significant natural abiotic stresses that occur in the intertidal zones. Seaweeds may physiologically acclimate to changing osmolarity by altering their transcriptome. Here, we investigated the transcriptomic changes of Gracilaria changii (B. M. Xia et I. A. Abbott) I. A. Abbott, J. Zhang et B. M. Xia in response to hyper- and hypoosmotic stresses using a cDNA microarray approach. Microarray analysis revealed that 199 and 200 genes from ∼3,300 genes examined were up- and down-regulated by >2-fold in seaweed samples treated at 50 parts per thousand (ppt) artificial seawater (ASW) compared with those at 30 ppt ASW, respectively. The number of genes that were up- and down-regulated by >2-fold in seaweed samples treated at 10 ppt ASW compared with those at 30 ppt ASW were 154 and 187, respectively. A majority of these genes were only differentially expressed under hyper- or hypoosmotic conditions, whereas 67 transcripts were affected by both stresses. The findings of this study have shed light on the expression profiles of many transcripts during the acclimation of G. changii to hyperosmotic and hypoosmotic conditions. This information may assist in the prioritization of genes to be examined in future studies. PMID:27032354

The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptomeanalysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest. PMID:25590333

We assembled a de novo transcriptome of short-read Illumina RNA-Seq data generated from telencephalon and diencephalon tissue samples from the Kentish plover, Charadrius alexandrinus. This is a species of considerable interest in behavioural ecology for its highly variable mating system and parental behaviour, but it lacks genomic resources and is evolutionarily distant from the few available avian draft genome sequences. We assembled and identified over 21,000 transcript contigs with significant expression in our samples, showing high homology to exonic sequences in avian draft genomes. From these, we identified >31,000 high-quality SNPs and > 2500 simple sequence repeats (SSRs). We also analysed expression patterns in our data to identify potential candidate genes related to differences in male and female behaviour, identifying over 200 nonoverlapping putative autosomal transcripts that show significant expression differences between males and females. Gene ontology analysis revealed that female-biased transcripts were significantly enriched for cerebral functions related to learning, cognition and memory, and male-biased transcripts were mostly enriched for terms related to neural function such as neuron projection and synapses. This data set provides one of the first de novo transcriptome assemblies from non-normalized short-read next-generation data and outlines an effective strategy for measuring sequence and expression variability simultaneously without the aid of a reference genome. PMID:23551815

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is maintained in nature in a cycle involving a tick vector and a mammalian host. Adaptation to the diverse conditions of temperature, pH, oxygen tension and nutrient availability in these two environments requires the precise orchestration of gene expression. Over 25 microarray analyses relating to B. burgdorferi genomics and transcriptomics have been published. The majority of these studies has explored the global transcriptome under a variety of conditions and has contributed substantially to the current understanding of B. burgdorferi transcriptional regulation. In this review, we present a summary of these studies with particular focus on those that helped define the roles of transcriptional regulators in modulating gene expression in the tick and mammalian milieus. By performing comparative analysis of results derived from the published microarray expression profiling studies, we identified composite gene lists comprising differentially expressed genes in these two environments. Further, we explored the overlap between the regulatory circuits that function during the tick and mammalian phases of the enzootic cycle. Taken together, the data indicate that there is interplay among the distinct signaling pathways that function in feeding ticks and during adaptation to growth in the mammal. PMID:27600075

Acaryochloris species are a genus of cyanobacteria that utilize chlorophyll (chl) d as their primary chlorophyll molecule during oxygenic photosynthesis. Chl d allows Acaryochloris to harvest red-shifted light, which gives them the ability to live in filtered light environments that are depleted in visible light. Although genomes of multiple Acaryochloris species have been sequenced, their analysis has not revealed how chl d is synthesized. Here, we demonstrate that Acaryochloris sp. CCMEE 5410 cells undergo chlorosis by nitrogen depletion and exhibit robust regeneration of chl d by nitrogen repletion. We performed a time course RNA-Seq experiment to quantify global transcriptomic changes during chlorophyll recovery. We observed upregulation of numerous known chl biosynthesis genes and also identified an oxygenase gene with a similar transcriptional profile as these chl biosynthesis genes, suggesting its possible involvement in chl d biosynthesis. Moreover, our data suggest that multiple prochlorophyte chlorophyll-binding homologs are important during chlorophyll recovery, and light-independent chl synthesis genes are more dominant than the light-dependent gene at the transcription level. Transcriptomic characterization of this organism provides crucial clues toward mechanistic elucidation of chl d biosynthesis. PMID:27276888

Upon colonization in the host gastrointestinal tract, the enteric bacterial pathogen Campylobacter jejuni is exposed to a variety of signaling molecules including the catecholamine hormones epinephrine (Epi) and norepinephrine (NE). NE has been observed to stimulate the growth and potentially enhance the pathogenicity of C. jejuni. However, the underlying mechanisms are still largely unknown. In this study, both Epi and NE were also observed to promote C. jejuni growth in MEMα-based iron-restricted medium. Adhesion and invasion of Caco-2 cells by C. jejuni were also enhanced upon exposure to Epi or NE. To further examine the effect of Epi or NE on the pathobiology of C. jejuni, transcriptomic profiles were conducted for C. jejuni NCTC 11168 that was cultured in iron-restricted medium supplemented with Epi or NE. Compared to the genes expressed in the absence of the catecholamine hormones, 183 and 156 genes were differentially expressed in C. jejuni NCTC 11168 that was grown in the presence of Epi and NE, respectively. Of these differentially expressed genes, 102 genes were common for both Epi and NE treatments. The genes differentially expressed by Epi or NE are involved in diverse cellular functions including iron uptake, motility, virulence, oxidative stress response, nitrosative stress tolerance, enzyme metabolism, DNA repair and metabolism and ribosomal protein biosynthesis. The transcriptomeanalysis indicated that Epi and NE have similar effects on the gene expression of C. jejuni, and provided insights into the delicate interaction between C. jejuni and intestinal stress hormones in the host. PMID:26042101

Various morphological and cytological traits of oocytes and their surrounding cumulus cells may be used to select oocytes for assisted reproduction. However, even with careful selection, successful IVF and subsequent embryo development remain uncertain. The factors that ensure oocyte competence are unclear and other approaches to assessing developmental potential must be explored. With the constant development of the molecular toolbox, genomic/transcriptomicanalysis is becoming a more and more interesting approach to understand oocyte quality on the basis of RNA composition. Using bovine and mouse models as well as human oocytes of known developmental potential, various efforts are underway to characterize the mRNA profile of the competent oocyte using microarray technology. The proliferation of gene expression data sets raises new opportunities to identify the mechanisms involved in this complex phenotype, which should lead to improved techniques of assisted reproduction. Although several molecular markers of oocyte quality are known, translating these into cellular functions remains challenging, largely due to the poor correlation between mRNA level and protein synthesis. Unlike most somatic cells, the oocyte can store mRNA for days, with transcriptional activity remaining at a halt during the 4-5 days beginning before ovulation and ending with embryonic genome activation. This review provides an overview of the transcriptomic data obtained from oocytes of different quality as well as interesting avenues to explore in order to improve our understanding of oocyte competence. PMID:24233546

Several fenpropathrin-resistant predatory mites have been reported. However, the molecular mechanism of the resistance remains unknown. In the present study, the Neoseiulus barkeri (N. barkeri) transcriptome was generated using the Illumina sequencing platform, 34,211 unigenes were obtained, and 15,987 were manually annotated. After manual annotation, attentions were attracted to resistance-related genes, such as voltage-gated sodium channel (VGSC), cytochrome P450s (P450s), and glutathione S-transferases (GSTs). A polymorphism analysis detected two point mutations (E1233G and S1282G) in the linker region between VGSC domain II and III. In addition, 43 putative P450 genes and 10 putative GST genes were identified from the transcriptome. Among them, two P450 genes, NbCYP4EV2 and NbCYP4EZ1, and four GST genes, NbGSTd01, NbGSTd02, NbGSTd03 and NbGSTm03, were remarkably overexpressed 3.64–46.69-fold in the fenpropathrin resistant strain compared to that in the susceptible strain. These results suggest that fenpropathrin resistance in N. barkeri is a complex biological process involving many genetic changes and provide new insight into the N. barkeri resistance mechanism. PMID:27240349

Several fenpropathrin-resistant predatory mites have been reported. However, the molecular mechanism of the resistance remains unknown. In the present study, the Neoseiulus barkeri (N. barkeri) transcriptome was generated using the Illumina sequencing platform, 34,211 unigenes were obtained, and 15,987 were manually annotated. After manual annotation, attentions were attracted to resistance-related genes, such as voltage-gated sodium channel (VGSC), cytochrome P450s (P450s), and glutathione S-transferases (GSTs). A polymorphism analysis detected two point mutations (E1233G and S1282G) in the linker region between VGSC domain II and III. In addition, 43 putative P450 genes and 10 putative GST genes were identified from the transcriptome. Among them, two P450 genes, NbCYP4EV2 and NbCYP4EZ1, and four GST genes, NbGSTd01, NbGSTd02, NbGSTd03 and NbGSTm03, were remarkably overexpressed 3.64-46.69-fold in the fenpropathrin resistant strain compared to that in the susceptible strain. These results suggest that fenpropathrin resistance in N. barkeri is a complex biological process involving many genetic changes and provide new insight into the N. barkeri resistance mechanism. PMID:27240349

The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptomeanalysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest. PMID:25590333

The innate immune system of Drosophila is activated by ingestion of microorganisms. D. melanogaster breeds on fruits fermented by Saccharomyces cerevisiae, whereas D. virilis breeds on slime flux and decaying bark of tree housing a variety of bacteria, yeasts, and molds. In this study, it is shown that D. virilis has a higher resistance to oral infection of a species of filamentous fungi belonging to the genus Penicillium compared to D. melanogaster. In response to the fungal infection, a transcriptome profile of immune-related genes was considerably different between D. melanogaster and D. virilis: the genes encoding antifungal peptides, Drosomycin and Metchnikowin, were highly expressed in D. melanogaster whereas, the genes encoding Diptericin and Defensin were highly expressed in D. virilis. On the other hand, the immune-induced molecule (IM) genes showed contrary expression patterns between the two species: they were induced by the fungal infection in D. melanogaster but tended to be suppressed in D. virilis. Our transcriptomeanalysis also showed newly predicted immune-related genes in D. virilis. These results suggest that the innate immune system has been extensively differentiated during the evolution of these Drosophila species. PMID:24151578

The silver carp (Hypophthalmichthys molitrix) is among the most intensively pond-cultured fish species and is used in the wild to counteract water bloom in China. However, little genomic information is available for this species, especially regarding its ability to grow rapidly in water, even water contaminated with high concentrations of poisonous microcystin. In this study, we performed de novo transcriptome assembly and analysis of the 17.10 million short-read sequences produced by the Illumina paired-end sequencing technology. Using an improved multiple k-mer contig assembly method coupled with further scaffolding, 85 759 sequences were obtained. There were 23 044 sequences annotated with 3423 gene ontology terms for 104 196 term occurrences and the three corresponding organizing principles. A total of 38 200 assembled sequences were involved in 218 predicted Kyoto Encyclopedia of Genes and Genomes metabolic pathways. We also recovered 41 of 44 genes involved in the biosynthesis of glutathione. Of these, five genes were identified as experienced positive selection between silver carp and zebrafish, as determined by the likelihood ratio test. This report is the first annotated review of the silver carp transcriptome. These data will be of interest to researchers investigating the evolution and biological processes of the silver carp. This work also provides an archive for future studies of recent speciation and evolution of Cyprinidae fishes and can be used in comparative studies of other fishes. PMID:22279088

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae, is a devastating disease in cotton. To get more candidate genes related to wilt resistance, a normalized, full-length cDNA library was used to generate the transcriptome profile from a wilt-resistant Gossypium hirsutum variety (Zhongzhimian KV3) upon V. dahliae strain V991 infection. Total 3486 high-quality ESTs were focused from randomly selected 4000 clones, which included 3065 singletons and 421 contigs. To categorize these unigenes, they were compared to functional domain databases. Total 106 unigenes were found to be homologous to known defense-related genes. Among them, PR genes were the major group. Furthermore, knockdown of GhWRKY22, GhWRKY33, GhChitinase, GhCML, and GhDirigent resulted in increased susceptibility of resistant cotton to V. dahliae infection. The results of transcriptome profiles and virus induced gene silencing (VIGS) analysis laid a good foundation for further investigation of cotton resistance to wilt disease. PMID:27038549

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is maintained in nature in a cycle involving a tick vector and a mammalian host. Adaptation to the diverse conditions of temperature, pH, oxygen tension and nutrient availability in these two environments requires the precise orchestration of gene expression. Over 25 microarray analyses relating to B. burgdorferi genomics and transcriptomics have been published. The majority of these studies has explored the global transcriptome under a variety of conditions and has contributed substantially to the current understanding of B. burgdorferi transcriptional regulation. In this review, we present a summary of these studies with particular focus on those that helped define the roles of transcriptional regulators in modulating gene expression in the tick and mammalian milieus. By performing comparative analysis of results derived from the published microarray expression profiling studies, we identified composite gene lists comprising differentially expressed genes in these two environments. Further, we explored the overlap between the regulatory circuits that function during the tick and mammalian phases of the enzootic cycle. Taken together, the data indicate that there is interplay among the distinct signaling pathways that function in feeding ticks and during adaptation to growth in the mammal. PMID:27600075

The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, D-mannitol and saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity. - Highlights: • The zebrafish embryotoxicity test in combination with transcriptomics was used. • We explored two approaches of defining gene biomarkers for developmental toxicity. • Four compounds in concentration-response design were tested. • We identified commonly expressed individual genes as well as regulated gene pathways. • Both approaches seem suitable starting points for defining gene biomarkers.

Identifying specific effects of contaminants in a multi-stress field context remain a challenge in ecotoxicology. In this context, "omics" technologies, by allowing the simultaneous measurement of numerous biological endpoints, could help unravel the in situ toxicity of contaminants. In this study, wild Atlantic eels were sampled in 8 sites presenting a broad contamination gradient in France and Canada. The global hepatic transcriptome of animals was determined by RNA-Seq. In parallel, the contamination level of fish to 8 metals and 25 organic pollutants was determined. Factor analysis for multiple testing was used to identify genes that are most likely to be related to a single factor. Among the variables analyzed, arsenic (As), cadmium (Cd), lindane (γ-HCH) and the hepato-somatic index (HSI) were found to be the main factors affecting eel's transcriptome. Genes associated with As exposure were involved in the mechanisms that have been described during As vasculotoxicity in mammals. Genes correlated with Cd were involved in cell cycle and energy metabolism. For γ-HCH, genes were involved in lipolysis and cell growth. Genes associated with HSI were involved in protein, lipid and iron metabolisms. Our study proposes specific gene signatures of pollutants and their impacts in fish exposed to multi-stress conditions. PMID:25258179

Background The plant tolerance mechanisms to low temperature have been studied extensively in the model plant Arabidopsis at the transcriptional level. However, few studies were carried out in plants with strong inherited cold tolerance. Chorispora bungeana is a subnival alpine plant possessing strong cold tolerance mechanisms. To get a deeper insight into its cold tolerance mechanisms, the transcriptome profiles of chilling-treated C. bungeana seedlings were analyzed by Illumina deep-sequencing and compared with Arabidopsis. Results Two cDNA libraries constructed from mRNAs of control and chilling-treated seedlings were sequenced by Illumina technology. A total of 54,870 unigenes were obtained by de novo assembly, and 3,484 chilling up-regulated and 4,571 down-regulated unigenes were identified. The expressions of 18 out of top 20 up-regulated unigenes were confirmed by qPCR analysis. Functional network analysis of the up-regulated genes revealed some common biological processes, including cold responses, and molecular functions in C. bungeana and Arabidopsis responding to chilling. Karrikins were found as new plant growth regulators involved in chilling responses of C. bungeana and Arabidopsis. However, genes involved in cold acclimation were enriched in chilling up-regulated genes in Arabidopsis but not in C. bungeana. In addition, although transcription activations were stimulated in both C. bungeana and Arabidopsis, no CBF putative ortholog was up-regulated in C. bungeana while CBF2 and CBF3 were chilling up-regulated in Arabidopsis. On the other hand, up-regulated genes related to protein phosphorylation and auto-ubiquitination processes were over-represented in C. bungeana but not in Arabidopsis. Conclusions We conducted the first deep-sequencing transcriptome profiling and chilling stress regulatory network analysis of C. bungeana, a subnival alpine plant with inherited cold tolerance. Comparative transcriptomeanalysis suggests that cold acclimation is not

The industrially important food-yeast Candida utilis is a Crabtree effect-negative yeast used to produce valuable chemicals and recombinant proteins. In the present study, we conducted whole genome sequencing and phylogenetic analysis of C. utilis, which showed that this yeast diverged long before the formation of the CUG and Saccharomyces/Kluyveromyces clades. In addition, we performed comparative genome and transcriptome analyses using next-generation sequencing, which resulted in the identification of genes important for characteristic phenotypes of C. utilis such as those involved in nitrate assimilation, in addition to the gene encoding the functional hexose transporter. We also found that an antisense transcript of the alcohol dehydrogenase gene, which in silico analysis did not predict to be a functional gene, was transcribed in the stationary-phase, suggesting a novel system of repression of ethanol production. These findings should facilitate the development of more sophisticated systems for the production of useful reagents using C. utilis. PMID:22629373

Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar ‘Tortuosa’). De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs) and 36 different expressed miRNAs (DEMs). Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix. PMID:25275458

Papilla and skin are two important organs of the sea cucumber. Both tissues have ectodermic origin, but they are morphologically and functionally very different. In the present study, we performed comparative transcriptomeanalysis of the papilla and skin from the sea cucumber (Apostichopus japonicus) in order to identify and characterize gene expression profiles by using RNA-Seq technology. We generated 30.6 and 36.4 million clean reads from the papilla and skin and de novo assembled in 156,501 transcripts. The Gene Ontology (GO) analysis indicated that cell part, metabolic process and catalytic activity were the most abundant GO category in cell component, biological process and molecular funcation, respectively. Comparative transcriptomeanalysis between the papilla and skin allowed the identification of 1,059 differentially expressed genes, of which 739 genes were expressed at higher levels in papilla, while 320 were expressed at higher levels in skin. In addition, 236 differentially expressed unigenes were not annotated with any database, 160 of which were apparently expressed at higher levels in papilla, 76 were expressed at higher levels in skin. We identified a total of 288 papilla-specific genes, 171 skin-specific genes and 600 co-expressed genes. Also, 40 genes in papilla-specific were not annotated with any database, 2 in skin-specific. Development-related genes were also enriched, such as fibroblast growth factor, transforming growth factor-β, collagen-α2 and Integrin-α2, which may be related to the formation of the papilla and skin in sea cucumber. Further pathway analysis identified ten KEGG pathways that were differently enriched between the papilla and skin. The findings on expression profiles between two key organs of the sea cucumber should be valuable to reveal molecular mechanisms involved in the development of organs that are related but with morphological differences in the sea cucumber. PMID:26989617

Papilla and skin are two important organs of the sea cucumber. Both tissues have ectodermic origin, but they are morphologically and functionally very different. In the present study, we performed comparative transcriptomeanalysis of the papilla and skin from the sea cucumber (Apostichopus japonicus) in order to identify and characterize gene expression profiles by using RNA-Seq technology. We generated 30.6 and 36.4 million clean reads from the papilla and skin and de novo assembled in 156,501 transcripts. The Gene Ontology (GO) analysis indicated that cell part, metabolic process and catalytic activity were the most abundant GO category in cell component, biological process and molecular funcation, respectively. Comparative transcriptomeanalysis between the papilla and skin allowed the identification of 1,059 differentially expressed genes, of which 739 genes were expressed at higher levels in papilla, while 320 were expressed at higher levels in skin. In addition, 236 differentially expressed unigenes were not annotated with any database, 160 of which were apparently expressed at higher levels in papilla, 76 were expressed at higher levels in skin. We identified a total of 288 papilla-specific genes, 171 skin-specific genes and 600 co-expressed genes. Also, 40 genes in papilla-specific were not annotated with any database, 2 in skin-specific. Development-related genes were also enriched, such as fibroblast growth factor, transforming growth factor-β, collagen-α2 and Integrin-α2, which may be related to the formation of the papilla and skin in sea cucumber. Further pathway analysis identified ten KEGG pathways that were differently enriched between the papilla and skin. The findings on expression profiles between two key organs of the sea cucumber should be valuable to reveal molecular mechanisms involved in the development of organs that are related but with morphological differences in the sea cucumber. PMID:26989617

Nitrogen (N) is the most important macronutrient for plant growth and grain yields. For rice crops, nitrate and ammonium are the major N sources. To explore the genomic responses to ammonium supplements in rice roots, we used 17-day-old seedlings grown in the absence of external N that were then exposed to 0.5mM (NH4)2SO4 for 3h. Transcriptomic profiles were examined by microarray experiments. In all, 634 genes were up-regulated at least two-fold by the N-supplement when compared with expression in roots from untreated control plants. Gene Ontology (GO) enrichment analysis revealed that those upregulated genes are associated with 23 GO terms. Among them, metabolic processes for diverse amino acids (i.e., aspartate, threonine, tryptophan, glutamine, l-phenylalanine, and thiamin) as well as nitrogen compounds are highly over-represented, demonstrating that our selected genes are suitable for studying the N-response in roots. This enrichment analysis also indicated that nitrogen is closely linked to diverse transporter activities by primary metabolites, including proteins (amino acids), lipids, and carbohydrates, and is associated with carbohydrate catabolism and cell wall organization. Integration of results from omics analysis of metabolic pathways and transcriptome data using the MapMan tool suggested that the TCA cycle and pathway for mitochondrial electron transport are co-regulated when rice roots are exposed to ammonium. We also investigated the expression of N-responsive marker genes by performing a comparative analysis with root samples from plants grown under different NH4(+) treatments. The diverse responses to such treatment provide useful insight into the global changes related to the shift from an N-deficiency to an enhanced N-supply in rice, a model crop plant. PMID:27340859

Background Cordyceps militaris, an ascomycete caterpillar fungus, has been used as a traditional Chinese medicine for many years owing to its anticancer and immunomodulatory activities. Currently, artificial culturing of this beneficial fungus has been widely used and can meet the market, but systematic molecular studies on the developmental stages of cultured C. militaris at transcriptional and translational levels have not been determined. Methodology/Principal Findings We utilized high-throughput Illumina sequencing to obtain the transcriptomes of C. militaris mycelium and fruiting body. All clean reads were mapped to C. militaris genome and most of the reads showed perfect coverage. Alternative splicing and novel transcripts were predicted to enrich the database. Gene expression analysis revealed that 2,113 genes were up-regulated in mycelium and 599 in fruiting body. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to analyze the genes with expression differences. Moreover, the putative cordycepin metabolism difference between different developmental stages was studied. In addition, the proteome data of mycelium and fruiting body were obtained by one-dimensional gel electrophoresis (1-DGE) coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). 359 and 214 proteins were detected from mycelium and fruiting body respectively. GO, KEGG and Cluster of Orthologous Groups (COG) analysis were further conducted to better understand their difference. We analyzed the amounts of some noteworthy proteins in these two samples including lectin, superoxide dismutase, glycoside hydrolase and proteins involved in cordycepin metabolism, providing important information for further protein studies. Conclusions/Significance The results reveal the difference in gene expression between the mycelium and fruiting body of artificially cultivated C. militaris by transcriptome and proteome

The turf grass Sporobolus virginicus is halophyte and has high salinity tolerance. To investigate the molecular basis of its remarkable tolerance, we performed Illumina high-throughput RNA sequencing on roots and shoots of a S. virginicus genotype under normal and saline conditions. The 130 million short reads were assembled into 444,242 unigenes. A comparative analysis of the transcriptome with rice and Arabidopsis transcriptome revealed six turf grass-specific unigenes encoding transcription factors. Interestingly, all of them showed root specific expression and five of them encode bZIP type transcription factors. Another remarkable transcriptional feature of S. virginicus was activation of specific pathways under salinity stress. Pathway enrichment analysis suggested transcriptional activation of amino acid, pyruvate, and phospholipid metabolism. Up-regulation of several unigenes, previously shown to respond to salt stress in other halophytes was also observed. Gene Ontology enrichment analysis revealed that unigenes assigned as proteins in response to water stress, such as dehydrin and aquaporin, and transporters such as cation, amino acid, and citrate transporters, and H+-ATPase, were up-regulated in both shoots and roots under salinity. A correspondence analysis of the enriched pathways in turf grass cells, but not in rice cells, revealed two groups of unigenes similarly up-regulated in the turf grass in response to salt stress; one of the groups, showing excessive up-regulation under salinity, included unigenes homologos to salinity responsive genes in other halophytes. Thus, the present study identified candidate genes involved in salt tolerance of S. virginicus. This genetic resource should be valuable for understanding the mechanisms underlying high salt tolerance in S. virginicus. This information can also provide insight into salt tolerance in other halophytes. PMID:25954282

The rhizome of Atractylodes lancea is extensively used in the practice of Traditional Chinese Medicine because of its broad pharmacological activities. This study was designed to characterize the transcriptome profiling of the rhizome and leaf of Atractylodes lancea in an attempt to uncover the molecular mechanisms regulating rhizome formation and growth. Over 270 million clean reads were assembled into 92,366 unigenes, 58% of which are homologous with sequences in public protein databases (NR, Swiss-Prot, GO, and KEGG). Analysis of expression levels showed that genes involved in photosynthesis, stress response, and translation were the most abundant transcripts in the leaf, while transcripts involved in stress response, transcription regulation, translation, and metabolism were dominant in the rhizome. Tissue-specific gene analysis identified distinct gene families active in the leaf and rhizome. Differential gene expression analysis revealed a clear difference in gene expression pattern, identifying 1518 up-regulated genes and 3464 down-regulated genes in the rhizome compared with the leaf, including a series of genes related to signal transduction, primary and secondary metabolism. Transcription factor (TF) analysis identified 42 TF families, with 67 and 60 TFs up-regulated in the rhizome and leaf, respectively. A total of 104 unigenes were identified as candidates for regulating rhizome formation and development. These data offer an overview of the gene expression pattern of the rhizome and leaf and provide essential information for future studies on the molecular mechanisms of controlling rhizome formation and growth. The extensive transcriptome data generated in this study will be a valuable resource for further functional genomics studies of A. lancea. PMID:27066021

Background Understanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out. Results A whole transcriptomeanalysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage. Conclusion The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns

Background Phialocephala subalpina belongs to the Phialocephala fortinii s.l.–Acepphala applanata species complex (PAC) forming one of the major groups belonging to the dark septate endophytes (DSE). Depending on the strain, PAC was shown to form neutral to pathogenic associations with its host plant Picea abies. To understand PACs lifestyle we investigated the effect of presence/absence of Picea abies on the transcriptome of strain 6_70_1. Materials and Methods PAC strain 6_70_1 was grown in liquid Pachlewski media either induced by its host plant Picea abies or without host plant as a control. Mycelia were harvested in a time course (1, 2, 3, 4, 7, 11, 18 days) with and without induction by the host plant and the fungal transcriptome revealed by Illumina sequencing. Differential gene expression analysis over the time course comparing control and treatment at each time point using the ‘edgeR glm approach’ and a gene enrichment analysis using GO categories were performed. Results The three main functional groups within differentially expressed genes were ‘metabolism’, ‘transport’ and ‘cell rescue, defense and virulence’. Additionally, genes especially involved in iron metabolism could be detected by gene set enrichment analysis. Conclusion In conclusion, we found PAC strain 6_70_1 to be metabolically very active during colonization of its host plant Picea abies. A major shift in functional groups over the time course of this experiment could not be observed but GO categories which were found to be enriched showed different emphasis depending in the day post induction. PMID:26954682

Ovarian granulosa cell tumors (GCT) are hormonally-active neoplasms characterized, in the adult-subtype, by a mutation in the FOXL2 gene (C134W). They exhibit an indolent course with an unexplained propensity for late recurrence; ∼80% of patients with aggressive, advanced stage tumors die from their disease; aside from surgery, therapeutic options are limited. To identify the molecular basis of advanced stage disease we have used whole transcriptomeanalysis of FOXL2 C134W mutation positive adult (a)GCT to identify genes that are differentially expressed between early (stage 1) and advanced (stage 3) aGCT. Transcriptome profiles for early (n = 6) and stage 3 (n = 6) aGCT, and for the aGCT-derived KGN, cell line identified 24 genes whose expression significantly differs between the early and stage 3 aGCT. Of these, 16 were more abundantly expressed in the stage 3 aGCT and 8 were higher in the stage 1 tumors. These changes were further examined for the genes which showed the greatest fold change: the cytokine CXCL14, microfibrillar-associated protein 5, insulin-like 3 and desmin. Gene Set Enrichment Analysis identified overexpression of genes on chromosome 7p15 which includes the homeobox A gene locus. The analysis therefore identifies a small number of genes with clearly discriminate patterns of expression arguing that the clinicopathological-derived distinction of the tumor stage is robust, whilst confirming the relative homogeneity of expression for many genes across the cohort and hence of aGCT. The expression profiles do however identify several overexpressed genes in both stage 1 and/or stage 3 aGCT which warrant further study as possible therapeutic targets. PMID:26893359

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the serious rice pests because of its destructive feeding. The salivary glands of the WBPH play an important role in the feeding behaviour. Currently, however, very little is known about the salivary glands at the molecular level. We sequenced the salivary gland transcriptome (sialotranscripome) of adult WBPHs using the Illumina sequencing. A total of 65,595 transcripts and 51,842 unigenes were obtained from salivary glands. According to annotations against the Nr database, many of the unigenes identified were associated with the most studied enzymes in hemipteran saliva. In the present study, we identified 32 salivary protein genes from the WBPH sialotranscripome, which were categorized as those involved in sugar metabolism, detoxification, suppression of plant defense responses, immunity-related responses, general digestion, and other phytophagy processes. Tissue expression profiles analysis revealed that four of 32 salivary protein genes (multicopper oxidase 4, multicopper oxidase 6, carboxylesterase and uridine phosphorylase 1 isform X2) were primarily expressed in the salivary gland, suggesting that they played putative role in insect-rice interactions. 13 of 32 salivary protein genes were primarily expressed in gut, which might play putative role in digestive and detoxify mechanism. Development expression profiles analysis revealed that the expression level of 26 of 32 salivary protein genes had no significant difference, suggesting that they may play roles in every developmental stages of salivary gland of WBPH. The other six genes have a high expression level in the salivary gland of adult. 31 of 32 genes (except putative acetylcholinesterase 1) have no significant difference in male and female adult, suggesting that their expression level have no difference between sexes. This report analysis of the sialotranscripome for the WBPH, and the transcriptome provides a foundational

Gibberellins (GAs) regulate a wide range of important processes in plant growth and development, including photosynthesis. However, the mechanism by which GAs regulate photosynthesis remains to be understood. Here, we used multi-gene association to investigate the effect of genes in the GA-responsive pathway, as constructed by RNA sequencing, on photosynthesis, growth, and wood property traits, in a population of 435 Populus tomentosa By analyzing changes in the transcriptome following GA treatment, we identified many key photosynthetic genes, in agreement with the observed increase in measurements of photosynthesis. Regulatory motif enrichment analysis revealed that 37 differentially expressed genes related to photosynthesis shared two essential GA-related cis-regulatory elements, the GA response element and the pyrimidine box. Thus, we constructed a GA-responsive pathway consisting of 47 genes involved in regulating photosynthesis, including GID1, RGA, GID2, MYBGa, and 37 photosynthetic differentially expressed genes. Single nucleotide polymorphism (SNP)-based association analysis showed that 142 SNPs, representing 40 candidate genes in this pathway, were significantly associated with photosynthesis, growth, and wood property traits. Epistasis analysis uncovered interactions between 310 SNP-SNP pairs from 37 genes in this pathway, revealing possible genetic interactions. Moreover, a structural gene-gene matrix based on a time-course of transcript abundances provided a better understanding of the multi-gene pathway affecting photosynthesis. The results imply a functional role for these genes in mediating photosynthesis, growth, and wood properties, demonstrating the potential of combining transcriptome-based regulatory pathway construction and genetic association approaches to detect the complex genetic networks underlying quantitative traits. PMID:27091876

The large amounts of transcriptome data available for Arabidopsis thaliana make a compelling case for the need to generalize results across studies and extract the most robust and meaningful information possible from them. The results of various studies seeking to identify water stress-responsive genes only partially overlap. The aim of this work was to combine transcriptomic studies in a systematic way that identifies commonalities in response, taking into account variation among studies due to batch effects as well as sampling variation, while also identifying the effect of study-specific variables, such as the method of applying water stress, and the part of the plant the mRNA was extracted from. We used meta-analysis, the quantitative synthesis of independent research results, to summarize expression responses to water stress across studies, and meta-regression to model the contribution of covariates that may affect gene expression. We found that some genes with small but consistent differential responses become evident only when results are synthesized across experiments, and are missed in individual studies. We also identified genes with expression responses that are attributable to use of different plant parts and alternative methods for inducing water stress. Our results indicate that meta-analysis and meta-regression provide a powerful approach for identifying a robust gene set that is less sensitive to idiosyncratic results and for quantifying study characteristics that result in contrasting gene expression responses across studies. Combining meta-analysis with individual analyses may contribute to a richer understanding of the biology of water stress responses, and may prove valuable in other gene expression studies. PMID:26756945

Coccolithophores are ideal for studying genes responsible for biomineralization processes due to relatively small genome sizes, ability to grow in culture, and as a natural model system for measuring expression of calcification-related genes in two life stages. As the Emiliania huxleyi has several annotated calcification-related proteins, we have concentrated on analyzing its genes and promoter areas. Many recent studies have focused primarily on transcriptomeanalysis of E. huxleyi using nutrient-limited conditions to get more information about up-regulated genes involved in biomineralization and calcification processes. Although there are more than 100,000 EST sequences for E. huxleyi available from these projects in public databases, that data is often insufficient to identify the exact position of transcription start site (TSS) to perform precise analysis (nucleotide content, motif search) of core promoters and regulatory mechanisms in immediate flanking areas. ESTs are not ideal for these kinds of analyses because the standard technologies of producing 5' EST libraries do not guarantee that the exact 5' end of the transcript will be captured. To determine the extent and accurate positions of 5' ends of transcripts and therefore the positions of core promoters, Cap analysis of gene expression (CAGE) sequencing method was used for sequencing RNA of E. huxleyi in both stages, calcifying and non-calcifying. As an additional info, gene expression levels of RNA for 21 samples were retrieved with whole transcriptome shotgun sequencing (RNA-Seq). The collections of reads these methods produced were used to map and annotate genes on several samples and measure the RNA expression levels in different conditions. Although there are not much data available for close organisms, it is possible to compare these results with other species to find conserved regulatory mechanisms between genes related to calcification. Visualization tools allowing browsing of annotated genes

Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of one-third of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/M-associated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components. PMID:15908605

The goal of this research was to investigate whether there has been a whole-genome duplication (WGD) in the ancestry of Sphagnum (peatmoss) or the class Sphagnopsida, and to determine if the timing of any such duplication(s) and patterns of paralog retention could help explain the rapid radiation and current ecological dominance of peatmosses. RNA sequencing (RNA-seq) data were generated for nine taxa in Sphagnopsida (Bryophyta). Analyses of frequency plots for synonymous substitutions per synonymous site (Ks ) between paralogous gene pairs and reconciliation of 578 gene trees were conducted to assess evidence of large-scale or genome-wide duplication events in each transcriptome. Both Ks frequency plots and gene tree-based analyses indicate multiple duplication events in the history of the Sphagnopsida. The most recent WGD event predates divergence of Sphagnum from the two other genera of Sphagnopsida. Duplicate retention is highly variable across species, which might be best explained by local adaptation. Our analyses indicate that the last WGD could have been an important factor underlying the diversification of peatmosses and facilitated their rise to ecological dominance in peatlands. The timing of the duplication events and their significance in the evolutionary history of peat mosses are discussed. PMID:26900928

Background Wheat seed germination directly affects wheat yield and quality. Although transcriptome and proteome analyses during seed germination have been reported in some crop plant species, dynamic transcriptome characterization during wheat seed germination has not been conducted. We performed the first comprehensive dynamic transcriptomeanalysis during different seed germination stages of elite Chinese bread wheat cultivar Jimai 20 using the Affymetrix Wheat Genome Array. Results A total of 61,703 probe sets representing 51,411 transcripts were identified during the five seed germination stages of Jimai 20, of which 2,825 differential expression probe sets corresponding to 2,646 transcripts with different functions were declared by ANOVA and a randomized variance model. The seed germination process included a rapid initial uptake phase (0–12 hours after imbibition [HAI]), a plateau phase (12–24 HAI), and a further water uptake phase (24–48 HAI), corresponding to switches from the degradation of small-molecule sucrose to the metabolism of three major nutrients and to photosynthesis. Hierarchical cluster and MapMan analyses revealed changes in several significant metabolism pathways during seed germination as well as related functional groups. The signal pathway networks constructed with KEGG showed three important genes encoding the phosphofructokinase family protein, with fructose-1, 6-bisphosphatase, and UTP-glucose-1-phosphate uridylyltransferase located at the center, indicating their pivotal roles in the glycolytic pathway, gluconeogenesis, and glycogenesis, respectively. Several significant pathways were selected to establish a metabolic pathway network according to their degree value, which allowed us to find the pathways vital to seed germination. Furthermore, 51 genes involved in transport, signaling pathway, development, lipid metabolism, defense response, nitrogen metabolism, and transcription regulation were analyzed by gene co

Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. Conclusion: A

Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production. PMID:16495679

Lactobacillus casei is a lactic acid bacterium commonly found in the gastrointestinal tract of animals, and some strains are used as probiotics. The ability of probiotic strains to survive the passage through the gastrointestinal tract is considered a key factor for their probiotic action. Therefore, tolerance to bile salts is a desirable feature for probiotic strains. In this study we have characterized the response of L. casei BL23 to bile by a transcriptomic and proteomic approach. The analysis revealed that exposure to bile induced changes in the abundance of 52 proteins and the transcript levels of 67 genes. The observed changes affected genes and proteins involved in the stress response, fatty acid and cell wall biosynthesis, metabolism of carbohydrates, transport of peptides, coenzyme levels, membrane H(+)-ATPase, and a number of uncharacterized genes and proteins. These data provide new insights into the mechanisms that enable L. casei BL23 to cope with bile stress. PMID:22322960

ABSTRACT Treatment of vulvovaginal candidiasis (VVC), caused most frequently by Candida albicans, represents a significant unmet clinical need. C. albicans, as both a commensal and a pathogenic organism, has a complex and poorly understood interaction with the vaginal environment. Understanding the complex nature of this relationship is necessary for the development of desperately needed therapies to treat symptomatic infection. Using transcriptome sequencing (RNA-seq), we characterized the early murine vaginal and fungal transcriptomes of the organism during VVC. Network analysis of host genes that were differentially expressed between infected and naive mice predicted the activation or repression of several signaling pathways that have not been previously associated with VVC, including NLRP3 inflammasome activation. Intravaginal challenge of Nlrp3−/− mice with C. albicans demonstrated severely reduced levels of polymorphonuclear leukocytes (PMNs), alarmins, and inflammatory cytokines, including interleukin-1β (IL-1β) (the hallmarks of VVC immunopathogenesis) in vaginal lavage fluid. Intravaginal administration of wild-type (WT) mice with glyburide, a potent inhibitor of the NLRP3 inflammasome, reduced PMN infiltration and IL-1β to levels comparable to those observed in Nlrp3−/− mice. Furthermore, RNA-seq analysis of C. albicans genes indicated robust expression of hypha-associated secreted aspartyl proteinases 4, 5, and 6 (SAP4–6), which are known inflammasome activators. Despite colonization similar to that of the WT strain, ΔSAP4–6 triple and ΔSAP5 single mutants induced significantly less PMN influx and IL-1β during intravaginal challenge. Our findings demonstrate a novel role for the inflammasome in the immunopathogenesis of VVC and implicate the hypha-associated SAPs as major C. albicans virulence determinants during vulvovaginal candidiasis. PMID:25900651

Cancer genomic studies that rely on analysis of biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in 9 hyperplastic ganglia harvested at 3 time points, and 4 tumor cohorts of progressively larger size in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We prioritized 3 targets (Cenpe, Gpr49, Impdh2) with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell lines, we further prioritized CENPE due to inhibition of cellular proliferation. Targeting CENPE with the small molecular inhibitor GSK923295 showed inhibition of in vitro proliferation of 19 neuroblastoma cell lines (median IC50=41 nM; range 27–266 nM), and delayed tumor growth in 3 xenograft models (p-values ranged from p<0.0001 to p=0.018). We provide preclinical validation that serial transcriptomeanalysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets, and identify CENPE as a novel therapeutic candidate in neuroblastoma. PMID:20233875

Background The common carp is an important aquaculture species that is widely distributed across the world. During the long history of carp domestication, numerous carp strains with diverse skin colors have been established. Skin color is used as a visual criterion to determine the market value of carp. However, the genetic basis of common carp skin color has not been extensively studied. Methodology/Principal Findings In this study, we performed Illumina sequencing on two common carp strains: the reddish Xingguo red carp and the brownish-black Yellow River carp. A total of 435,348,868 reads were generated, resulting in 198,781 assembled contigs that were used as reference sequences. Comparisons of skin transcriptome files revealed 2,012 unigenes with significantly different expression in the two common carp strains, including 874 genes that were up-regulated in Xingguo red carp and 1,138 genes that were up-regulated in Yellow River carp. The expression patterns of 20 randomly selected differentially expressed genes were validated using quantitative RT-PCR. Gene pathway analysis of the differentially expressed genes indicated that melanin biosynthesis, along with the Wnt and MAPK signaling pathways, is highly likely to affect the skin pigmentation process. Several key genes involved in the skin pigmentation process, including TYRP1, SILV, ASIP and xCT, showed significant differences in their expression patterns between the two strains. Conclusions In this study, we conducted a comparative transcriptomeanalysis of Xingguo red carp and Yellow River carp skins, and we detected key genes involved in the common carp skin pigmentation process. We propose that common carp skin pigmentation depends upon at least three pathways. Understanding fish skin color genetics will facilitate future molecular selection of the fish skin colors with high market values. PMID:25255374

The aim of this study was to identify pathways that have a significant impact during renal carcinogenesis. Sixty-seven paired samples of both noncancerous renal cortex tissue and cancerous tissue from patients with clear cell renal cell carcinomas (RCCs) were subjected to whole-exome, methylome and transcriptome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Illumina Infinium HumanMethylation27 BeadArray and Agilent SurePrint Human Gene Expression microarray, respectively. Sanger sequencing and quantitative reverse transcription-PCR were performed for technical verification. MetaCore software was used for pathway analysis. Somatic nonsynonymous single-nucleotide mutations, insertions/deletions and intragenic breaks of 2,153, 359 and 8 genes were detected, respectively. Mutations of GCN1L1, MED12 and CCNC, which are members of CDK8 mediator complex directly regulating β-catenin-driven transcription, were identified in 16% of the RCCs. Mutations of MACF1, which functions in the Wnt/β-catenin signaling pathway, were identified in 4% of the RCCs. A combination of methylome and transcriptome analyses further highlighted the significant role of the Wnt/β-catenin signaling pathway in renal carcinogenesis. Genetic aberrations and reduced expression of ERC2 and ABCA13 were frequent in RCCs, and MTOR mutations were identified as one of the major disrupters of cell signaling during renal carcinogenesis. Our results confirm that multilayer-omics analysis can be a powerful tool for revealing pathways that play a significant role in carcinogenesis. PMID:24504440

Monoecious species provide a comprehensive system to study the developmental programs underlying the establishment of female and male organs in unisexual flowers. However, molecular resources for most monoecious non-model species are limited, hampering our ability to study the molecular mechanisms involved in flower development of these species. The objective of this study was to identify differentially expressed genes during the development of male and female flowers of the monoecious species Quercus suber, an economically important Mediterranean tree. Total RNA was extracted from different developmental stages of Q. suber flowers. Non-normalized cDNA libraries of male and female flowers were generated using 454 pyrosequencing technology producing a total of 962,172 high-quality reads with an average length of 264 nucleotides. The assembly of the reads resulted in 14,488 contigs for female libraries and 10,438 contigs for male libraries. Comparative analysis of the transcriptomes revealed genes differentially expressed in early and late stages of development of female and male flowers, some of which have been shown to be involved in pollen development, in ovule formation and in flower development of other species with a monoecious, dioecious, or hermaphroditic sexual system. Moreover, we found differentially expressed genes that have not yet been characterized and others that have not been previously shown to be implicated in flower development. This transcriptomicanalysis constitutes a major step toward the characterization of the molecular mechanisms involved in flower development in a monoecious tree with a potential contribution toward the knowledge of conserved developmental mechanisms in other species. PMID:25414713

Peripheral glia are known to have a critical role in the initial response to axon damage and degeneration. However, little is known about the cellular responses of non-myelinating glia to nerve injury. In this study, we analyzed the transcriptomes of wild-type and mutant (lacking peripheral glia) zebrafish larvae that were treated with metronidazole. This treatment allowed us to conditionally and selectively ablate cranial sensory neurons whose axons are ensheathed only by non-myelinating glia. While transcripts representing over 27,000 genes were detected by RNAseq, only a small fraction (~1% of genes) were found to be differentially expressed in response to neuronal degeneration in either line at either 2 hrs or 5 hrs of metronidazole treatment. Analysis revealed that most expression changes (332 out of the total of 458 differentially expressed genes) occurred over a continuous period (from 2 to 5 hrs of metronidazole exposure), with a small number of genes showing changes limited to only the 2 hr (55 genes) or 5 hr (71 genes) time points. For genes with continuous alterations in expression, some of the most meaningful sets of enriched categories in the wild-type line were those involving the inflammatory TNF-alpha and IL6 signaling pathways, oxidoreductase activities and response to stress. Intriguingly, these changes were not observed in the mutant line. Indeed, cluster analysis indicated that the effects of metronidazole treatment on gene expression was heavily influenced by the presence or absence of glia, indicating that the peripheral non-myelinating glia play a significant role in the transcriptional response to sensory neuron degeneration. This is the first transcriptome study of metronidazole-induced neuronal death in zebrafish and the response of non-myelinating glia to sensory neuron degeneration. We believe this study provides important insight into the mechanisms by which non-myelinating glia react to neuronal death and degeneration in sensory

Despite constituting approximately two thirds of all breast cancers, the luminal A and B tumours are poorly classified at both clinical and molecular levels. There are contradictory reports on the nature of these subtypes: some define them as intrinsic entities, others as a continuum. With the aim of addressing these uncertainties and identifying molecular signatures of patients at risk, we conducted a comprehensive transcriptomic and genomic analysis of 2,425 luminal breast cancer samples. Our results indicate that the separation between the molecular luminal A and B subtypes—per definition—is not associated with intrinsic characteristics evident in the differentiation between other subtypes. Moreover, t-SNE and MST-kNN clustering approaches based on 10,000 probes, associated with luminal tumour initiation and/or development, revealed the close connections between luminal A and B tumours, with no evidence of a clear boundary between them. Thus, we considered all luminal tumours as a single heterogeneous group for analysis purposes. We first stratified luminal tumours into two distinct groups by their HER2 gene cluster co-expression: HER2-amplified luminal and ordinary-luminal. The former group is associated with distinct transcriptomic and genomic profiles, and poor prognosis; it comprises approximately 8% of all luminal cases. For the remaining ordinary-luminal tumours we further identified the molecular signature correlated with disease outcomes, exhibiting an approximately continuous gene expression range from low to high risk. Thus, we employed four virtual quantiles to segregate the groups of patients. The clinico-pathological characteristics and ratios of genomic aberrations are concordant with the variations in gene expression profiles, hinting at a progressive staging. The comparison with the current separation into luminal A and B subtypes revealed a substantially improved survival stratification. Concluding, we suggest a review of the definition of

Tissue culture could solve the problems associated with Gracilaria cultivation, including the consistent supply of high-quality seed stock, strain improvement, and efficient mass culture of high-yielding commercial strains. However, STC lags behind that of higher plants because of the paucity of genomic information. Transcriptomeanalysis and the identification of potential unigenes involved in the formation and regeneration of callus or direct induction of ABs are essential. Herein, the CK, EWAB and NPA G. lichenoides transcriptomes were analyzed using the Illumina sequencing platform in first time. A total of 17,922,453,300 nucleotide clean bases were generated and assembled into 21,294 unigenes, providing a total gene space of 400,912,038 nucleotides with an average length of 1,883 and N 50 of 5,055 nucleotides and a G + C content of 52.02%. BLAST analysis resulted in the assignment of 13,724 (97.5%), 3,740 (26.6%), 9,934 (70.6%), 10,611 (75.4%), 9,490 (67.4%), and 7,773 (55.2%) unigenes were annotated to the NR, NT, Swiss-Prot, KEGG, COG, and GO databases, respectively, and the total of annotated unigenes was 14,070. A total of 17,099 transcripts were predicted to possess open reading frames, including 3,238 predicted and 13,861 blasted based on protein databases. In addition, 3,287 SSRs were detected in G.lichenoides, providing further support for genetic variation and marker-assisted selection in the future. Our results suggest that auxin polar transport, auxin signal transduction, crosstalk with other endogenous plant hormones and antioxidant systems, play important roles for ABs formation in G. lichenoides explants in vitro. The present findings will facilitate further studies on gene discovery and on the molecular mechanisms underlying the tissue culture of seaweed. PMID:26657019

Despite constituting approximately two thirds of all breast cancers, the luminal A and B tumours are poorly classified at both clinical and molecular levels. There are contradictory reports on the nature of these subtypes: some define them as intrinsic entities, others as a continuum. With the aim of addressing these uncertainties and identifying molecular signatures of patients at risk, we conducted a comprehensive transcriptomic and genomic analysis of 2,425 luminal breast cancer samples. Our results indicate that the separation between the molecular luminal A and B subtypes-per definition-is not associated with intrinsic characteristics evident in the differentiation between other subtypes. Moreover, t-SNE and MST-kNN clustering approaches based on 10,000 probes, associated with luminal tumour initiation and/or development, revealed the close connections between luminal A and B tumours, with no evidence of a clear boundary between them. Thus, we considered all luminal tumours as a single heterogeneous group for analysis purposes. We first stratified luminal tumours into two distinct groups by their HER2 gene cluster co-expression: HER2-amplified luminal and ordinary-luminal. The former group is associated with distinct transcriptomic and genomic profiles, and poor prognosis; it comprises approximately 8% of all luminal cases. For the remaining ordinary-luminal tumours we further identified the molecular signature correlated with disease outcomes, exhibiting an approximately continuous gene expression range from low to high risk. Thus, we employed four virtual quantiles to segregate the groups of patients. The clinico-pathological characteristics and ratios of genomic aberrations are concordant with the variations in gene expression profiles, hinting at a progressive staging. The comparison with the current separation into luminal A and B subtypes revealed a substantially improved survival stratification. Concluding, we suggest a review of the definition of

Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ∼3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptomeanalysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptomeanalysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity. PMID:21317319

Tissue culture could solve the problems associated with Gracilaria cultivation, including the consistent supply of high-quality seed stock, strain improvement, and efficient mass culture of high-yielding commercial strains. However, STC lags behind that of higher plants because of the paucity of genomic information. Transcriptomeanalysis and the identification of potential unigenes involved in the formation and regeneration of callus or direct induction of ABs are essential. Herein, the CK, EWAB and NPA G. lichenoides transcriptomes were analyzed using the Illumina sequencing platform in first time. A total of 17,922,453,300 nucleotide clean bases were generated and assembled into 21,294 unigenes, providing a total gene space of 400,912,038 nucleotides with an average length of 1,883 and N 50 of 5,055 nucleotides and a G + C content of 52.02%. BLAST analysis resulted in the assignment of 13,724 (97.5%), 3,740 (26.6%), 9,934 (70.6%), 10,611 (75.4%), 9,490 (67.4%), and 7,773 (55.2%) unigenes were annotated to the NR, NT, Swiss-Prot, KEGG, COG, and GO databases, respectively, and the total of annotated unigenes was 14,070. A total of 17,099 transcripts were predicted to possess open reading frames, including 3,238 predicted and 13,861 blasted based on protein databases. In addition, 3,287 SSRs were detected in G.lichenoides, providing further support for genetic variation and marker-assisted selection in the future. Our results suggest that auxin polar transport, auxin signal transduction, crosstalk with other endogenous plant hormones and antioxidant systems, play important roles for ABs formation in G. lichenoides explants in vitro. The present findings will facilitate further studies on gene discovery and on the molecular mechanisms underlying the tissue culture of seaweed. PMID:26657019

Organohalide respiration, mediated by Dehalococcoides mccartyi, is a useful bioremediation process that transforms ground water pollutants and known human carcinogens such as trichloroethene and vinyl chloride into benign ethenes. Successful application of this process depends on the fundamental understanding of the respiration and metabolism of D. mccartyi. Reductive dehalogenases, encoded by rdhA genes of these anaerobic bacteria, exclusively catalyze organohalide respiration and drive metabolism. To better elucidate D. mccartyi metabolism and physiology, we analyzed available transcriptomic data for a pure isolate (Dehalococcoides mccartyi strain 195) and a mixed microbial consortium (KB-1) using the previously developed pan-genome-scale reconstructed metabolic network of D. mccartyi. The transcriptomic data, together with available proteomic data helped confirm transcription and expression of the majority genes in D. mccartyi genomes. A composite genome of two highly similar D. mccartyi strains (KB-1 Dhc) from the KB-1 metagenome sequence was constructed, and operon prediction was conducted for this composite genome and other single genomes. This operon analysis, together with the quality threshold clustering analysis of transcriptomic data helped generate experimentally testable hypotheses regarding the function of a number of hypothetical proteins and the poorly understood mechanism of energy conservation in D. mccartyi. We also identified functionally enriched important clusters (13 for strain 195 and 11 for KB-1 Dhc) of co-expressed metabolic genes using information from the reconstructed metabolic network. This analysis highlighted some metabolic genes and processes, including lipid metabolism, energy metabolism, and transport that potentially play important roles in organohalide respiration. Overall, this study shows the importance of an organism's metabolic reconstruction in analyzing various "omics" data to obtain improved understanding of the

Organohalide respiration, mediated by Dehalococcoides mccartyi, is a useful bioremediation process that transforms ground water pollutants and known human carcinogens such as trichloroethene and vinyl chloride into benign ethenes. Successful application of this process depends on the fundamental understanding of the respiration and metabolism of D. mccartyi. Reductive dehalogenases, encoded by rdhA genes of these anaerobic bacteria, exclusively catalyze organohalide respiration and drive metabolism. To better elucidate D. mccartyi metabolism and physiology, we analyzed available transcriptomic data for a pure isolate (Dehalococcoides mccartyi strain 195) and a mixed microbial consortium (KB-1) using the previously developed pan-genome-scale reconstructed metabolic network of D. mccartyi. The transcriptomic data, together with available proteomic data helped confirm transcription and expression of the majority genes in D. mccartyi genomes. A composite genome of two highly similar D. mccartyi strains (KB-1 Dhc) from the KB-1 metagenome sequence was constructed, and operon prediction was conducted for this composite genome and other single genomes. This operon analysis, together with the quality threshold clustering analysis of transcriptomic data helped generate experimentally testable hypotheses regarding the function of a number of hypothetical proteins and the poorly understood mechanism of energy conservation in D. mccartyi. We also identified functionally enriched important clusters (13 for strain 195 and 11 for KB-1 Dhc) of co-expressed metabolic genes using information from the reconstructed metabolic network. This analysis highlighted some metabolic genes and processes, including lipid metabolism, energy metabolism, and transport that potentially play important roles in organohalide respiration. Overall, this study shows the importance of an organism's metabolic reconstruction in analyzing various “omics” data to obtain improved understanding of the

Fish gills represent a complex organ composed of several cell types that perform multiple physiological functions. Among these cells, ionocytes are implicated in the maintenance of ion homeostasis. However, because the ionocyte represents only a small percent of whole gill tissue, its specific transcriptome can be overlooked among the numerous cell types included in the gill. The objective of this study is to better understand ionocyte functions by comparing the RNA expression of this cell type in freshwater and seawater acclimated rainbow trout. To realize this objective, ionocytes were captured from gill cryosections using laser capture microdissection after immunohistochemistry. Then, transcriptome analyses were performed on an Agilent trout oligonucleotide microarray. Gene expression analysis identified 108 unique annotated genes differentially expressed between freshwater and seawater ionocytes, with a fold change higher than 3. Most of these genes were up-regulated in freshwater cells. Interestingly, several genes implicated in ion transport, extracellular matrix and structural cellular proteins appeared up-regulated in freshwater ionocytes. Among them, several ion transporters, such as CIC2, SLC26A6, and NBC, were validated by qPCR and/or in situ hybridization. The latter technique allowed us to localize the transcripts of these ion transporters in only ionocytes and more particularly in the freshwater cells. Genes involved in metabolism and also several genes implicated in transcriptional regulation, cell signaling and the cell cycle were also enhanced in freshwater ionocytes. In conclusion, laser capture microdissection combined with microarray analysis allowed for the determination of the transcriptional signature of scarce cells in fish gills, such as ionocytes, and aided characterization of the transcriptome of these cells in freshwater and seawater acclimated trout. PMID:26439495

Purpose. To decipher transcriptomic changes and related genes with potential functions against Bombyx mori nucleopolyhedrovirus infection and to increase the understanding of the enhanced virus resistance of silkworm on the transcriptomic level. Methods. We assembled and annotated transcriptomes of the Qiufeng (susceptible to infection) and QiufengN (resistant to infection) strains and performed comparative analysis in order to decipher transcriptomic changes and related genes with potential functions against BmNPV infection. Results. A total of 78,408 SNPs were identified in the Qiufeng strain of silkworm and 56,786 SNPs were identified in QiufengN strain. Besides, novel AS events were found in these 2 strains. In addition, 1,728 DEGs were identified in the QiufengN strain compared with Qiufeng strain. These DEGs were involved in GO terms related to membrane, metabolism, binding and catalytic activity, cellular processes, and organismal systems. The highest levels of gene representation were found in oxidative phosphorylation, phagosome, TCA cycle, arginine and proline metabolism, and pyruvate metabolism. Additionally, COG analysis indicated that DEGs were involved in "amino acid transport and metabolism" and "carbohydrate transport and metabolism." Conclusion. We identified a series of major pathological changes in silkworm following infection and several functions were related to the antiviral mechanisms of silkworm. PMID:27195279

Recently, we found dramatic mitochondrial DNA divergence of Israeli Chamaeleo chamaeleon populations into two geographically distinct groups. We aimed to examine whether the same pattern of divergence could be found in nuclear genes. However, no genomic resource is available for any chameleon species. Here we present the first chameleon transcriptome, obtained using deep sequencing (SOLiD). Our analysis identified 164,000 sequence contigs of which 19,000 yielded unique BlastX hits. To test the efficacy of our sequencing effort, we examined whether the chameleon and other available reptilian transcriptomes harbored complete sets of genes comprising known biochemical pathways, focusing on the nDNA-encoded oxidative phosphorylation (OXPHOS) genes as a model. As a reference for the screen, we used the human 86 (including isoforms) known structural nDNA-encoded OXPHOS subunits. Analysis of 34 publicly available vertebrate transcriptomes revealed orthologs for most human OXPHOS genes. However, OXPHOS subunit COX8 (Cytochrome C oxidase subunit 8), including all its known isoforms, was consistently absent in transcriptomes of iguanian lizards, implying loss of this subunit during the radiation of this suborder. The lack of COX8 in the suborder Iguania is intriguing, since it is important for cellular respiration and ATP production. Our sequencing effort added a new resource for comparative genomic studies, and shed new light on the evolutionary dynamics of the OXPHOS system. PMID:24009133

Purpose. To decipher transcriptomic changes and related genes with potential functions against Bombyx mori nucleopolyhedrovirus infection and to increase the understanding of the enhanced virus resistance of silkworm on the transcriptomic level. Methods. We assembled and annotated transcriptomes of the Qiufeng (susceptible to infection) and QiufengN (resistant to infection) strains and performed comparative analysis in order to decipher transcriptomic changes and related genes with potential functions against BmNPV infection. Results. A total of 78,408 SNPs were identified in the Qiufeng strain of silkworm and 56,786 SNPs were identified in QiufengN strain. Besides, novel AS events were found in these 2 strains. In addition, 1,728 DEGs were identified in the QiufengN strain compared with Qiufeng strain. These DEGs were involved in GO terms related to membrane, metabolism, binding and catalytic activity, cellular processes, and organismal systems. The highest levels of gene representation were found in oxidative phosphorylation, phagosome, TCA cycle, arginine and proline metabolism, and pyruvate metabolism. Additionally, COG analysis indicated that DEGs were involved in “amino acid transport and metabolism” and “carbohydrate transport and metabolism.” Conclusion. We identified a series of major pathological changes in silkworm following infection and several functions were related to the antiviral mechanisms of silkworm. PMID:27195279

Previous studies have investigated the physiological responses to chronic copper (Cu) exposure in the liver of Synechogobius hasta; however, little information is available on the underlying molecular mechanisms. In an effort to better understand the mechanisms of Cu toxicity and to illuminate global gene expression patterns modulated by Cu exposure, we obtained the liver transcriptome information of S. hasta by RNA sequencing (RNA-seq) technology and also investigated the differential expression of genes following waterborne Cu exposure. Using the Illumina sequencing platform, as many as 60,217 unigenes were generated, with 815 bp of average length and 1298 bp of unigene N50 after filtering and assembly. For functional annotation analysis, 34,860, 31,526, 31,576, 25,808, 11,542, and 21,721 unigenes were annotated to the NR, NT, Swiss-Prot, KEGG, COG, and GO databases, respectively, and total annotation unigenes were 37,764. After 30 days of exposure to 55 μg Cu/l, a total of 292 and 1076 genes were significantly up- and down-regulated, respectively. By KEGG analysis, 660 had a specific pathway annotation. Subsequent bioinformatics analysis revealed that the differentially expressed genes were mainly related to lipid metabolism, immune system, apoptosis, and signal transduction, suggesting that these signaling pathways may be regulated by Cu exposure. The present study provides comprehensive sequence information for subsequent gene expression studies regarding S. hasta, and the transcriptome profiling after Cu exposure is also expected to improve our understanding of the molecular toxicology of Cu. PMID:26719065

Most higher plants are unable to survive desiccation to an air-dried state. An exception is a small group of vascular angiosperm plants, termed resurrection plants. They have evolved unique mechanisms of desiccation tolerance and thus can tolerate severe water loss, and mostly adjust their water content with the relative humidity in the environment. Desiccation tolerance is a complex phenomenon and depends on the regulated expression of numerous genes during dehydration and subsequent rehydration. Most of the resurrection plants have a large genome and are difficult to transform which makes them unsuitable for genetic approaches. However, technical advances have made it possible to analyze changes in gene expression on a large-scale. These approaches together with comparative studies with non-desiccation tolerant plants provide novel insights into the molecular processes required for desiccation tolerance and will shed light on identification of orphan genes with unknown functions. Here, we review large-scale recent transcriptomic, proteomic, and metabolomic studies that have been performed in desiccation tolerant plants and discuss how these studies contribute to understanding the molecular basis of desiccation tolerance. PMID:24348488

Using the Eimeria spp. population that infect chickens as a model for coccidian biology, we aimed to survey the transcriptome of E. maxima and contrast it to the two other Eimeria spp. for which transcriptome data are available, E. tenella and E. acervulina. Examining specifically the asexual intra...

Anthurium is one of the most important tropical ornamental plants in the world. The traded value of anthurium is second only to that of tropical orchids among the tropical flowers. The spathe is the main ornamental organ and its color variation mainly arises from anthocyanin contents. Understanding the molecular regulation of spathe color will accelerate new variety creation of anthurium. To announce gene expression differences between Anthurium andraeanum ‘Albama’ and its one unique anthocyanin-loss mutant, we collected spathes of the wild-type and the mutant from two stages in spathe development (the flower separates protrude from the sheath and the spathe is fully expanded) and extracted total RNAs for transcriptome profiling. Using short read sequencing technology (Illumina), 51,955,564, 53,822,224, 54,221,990 and 52,276,418 sequencing raw reads, respectively, for wild-type and mutant in the two stages were assembled de novo into 111,268 unique sequences (unigenes) with a mean length of 652 bp. 47,563 unigenes had significant hits to the sequences in the Nr database, and 32,768 unigenes showed significant similarity to known proteins in the Swiss-Prot database. 28,350 and 19,293 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Further, analysis of differentially expressed genes in the comparison between wild-type and mutant and between the two different developmental stages was carried out, indicating that the expression of an extensive set of genes changed as the result of mutation. Taken together, these data demonstrated that the Illumina sequencing allowed de novo transcriptome assembly and could obtain differentially expressed genes between A. andraeanum wild-type and the anthocyanin-loss mutant. The expression differences of AN2 and UFGT might cause the anthocyanin-loss mutation. PMID:25781998

Triplophysa dalaica, endemic species of Qinghai-Tibetan Plateau, is informative for understanding the genetic basis of adaptation to hypoxic conditions of high altitude habitats. Here, a comprehensive gene repertoire for this plateau fish was generated using the Illumina deep paired-end high-throughput sequencing technology. De novo assembly yielded 145, 256 unigenes with an average length of 1632 bp. Blast searches against GenBank non-redundant database annotated 74,594 (51.4%) unigenes encoding for 30,047 gene descriptions in T. dalaica. Functional annotation and classification of assembled sequences were performed using Gene Ontology (GO), clusters of euKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. After comparison with other fish transcriptomes, including silver carp (Hypophthalmichthys molitrix) and mud loach (Misgurnus anguillicaudatus), 2621 high-quality orthologous gene alignments were constructed among these species. 61 (2.3%) of the genes were identified as having undergone positive selection in the T. dalaica lineage. Within the positively selected genes, 13 genes were involved in hypoxia response, of which 11 were listed in HypoxiaDB. Furthermore, duplicated hif-α (hif-1αA/B and hif-2αA/B), EGLN1 and PPARA candidate genes involved in adaptation to hypoxia were identified in T. dalaica transcriptome. Branch-site model in PAML validated that hif-1αB and hif-2αA genes have undergone positive selection in T.dalaica. Finally, 37,501 simple sequence repeats (SSRs) and 19,497 high-quality single nucleotide polymorphisms (SNPs) were identified in T. dalaica. The identified SSR and SNP markers will facilitate the genetic structure, population geography and ecological studies of Triplophysa fishes. PMID:25869933

Background Sacha Inchi (Plukenetia volubilis L., Euphorbiaceae) is a potential oilseed crop because the seeds of this plant are rich in unsaturated fatty acids (FAs). In particular, the fatty acid composition of its seed oil differs markedly in containing large quantities of α-linolenic acid (18C:3, a kind of ω-3 FAs). However, little is known about the molecular mechanisms responsible for biosynthesis of unsaturated fatty acids in the developing seeds of this species. Transcriptome data are needed to better understand these mechanisms. Results In this study, de novo transcriptome assembly and gene expression analysis were performed using Illumina sequencing technology. A total of 52.6 million 90-bp paired-end reads were generated from two libraries constructed at the initial stage and fast oil accumulation stage of seed development. These reads were assembled into 70,392 unigenes; 22,179 unigenes showed a 2-fold or greater expression difference between the two libraries. Using this data we identified unigenes that may be involved in de novo FA and triacylglycerol biosynthesis. In particular, a number of unigenes encoding desaturase for formation of unsaturated fatty acids with high expression levels in the fast oil accumulation stage compared with the initial stage of seed development were identified. Conclusions This study provides the first comprehensive dataset characterizing Sacha Inchi gene expression at the transcriptional level. These data provide the foundation for further studies on molecular mechanisms underlying oil accumulation and PUFA biosynthesis in Sacha Inchi seeds. Our analyses facilitate understanding of the molecular mechanisms responsible for the high unsaturated fatty acids (especially α-linolenic acid) accumulation in Sacha Inchi seeds. PMID:23256450

Lathyrus sativus (grass pea) is a temperate grain legume crop with a great potential for expansion in dry areas or zones that are becoming more drought-prone. It is also recognized as a potential source of resistance to several important diseases in legumes, such as ascochyta blight. Nevertheless, the lack of detailed genomic and/or transcriptomic information hampers further exploitation of grass pea resistance-related genes in precision breeding. To elucidate the pathways differentially regulated during ascochyta-grass pea interaction and to identify resistance candidate genes, we compared the early response of the leaf gene expression profile of a resistant L. sativus genotype to Ascochyta lathyri infection with a non-inoculated control sample from the same genotype employing deepSuperSAGE. This analysis generated 14.387 UniTags of which 95.7% mapped to a reference grass pea/rust interaction transcriptome. From the total mapped UniTags, 738 were significantly differentially expressed between control and inoculated leaves. The results indicate that several gene classes acting in different phases of the plant/pathogen interaction are involved in the L. sativus response to A. lathyri infection. Most notably a clear up-regulation of defense-related genes involved in and/or regulated by the ethylene pathway was observed. There was also evidence of alterations in cell wall metabolism indicated by overexpression of cellulose synthase and lignin biosynthesis genes. This first genome-wide overview of the gene expression profile of the L. sativus response to ascochyta infection delivered a valuable set of candidate resistance genes for future use in precision breeding. PMID:25852725

Background Transcriptome variability is due to genetic and environmental causes, much like any other complex phenotype. Ascertaining the transcriptome differences between individuals is an important step to understand how selection and genetic drift may affect gene expression. To that end, extant divergent livestock breeds offer an ideal genetic material. Results We have analyzed with microarrays five tissues from the endocrine axis (hypothalamus, adenohypophysis, thyroid gland, gonads and fat tissue) of 16 pigs from both sexes pertaining to four extreme breeds (Duroc, Large White, Iberian and a cross with SinoEuropean hybrid line). Using a Bayesian linear model approach, we observed that the largest breed variability corresponded to the male gonads, and was larger than at the remaining tissues, including ovaries. Measurement of sex hormones in peripheral blood at slaughter did not detect any breed-related differences. Not unexpectedly, the gonads were the tissue with the largest number of sex biased genes. There was a strong correlation between sex and breed bias expression, although the most breed biased genes were not the most sex biased genes. A combined analysis of connectivity and differential expression suggested three biological processes as being primarily different between breeds: spermatogenesis, muscle differentiation and several metabolic processes. Conclusion These results suggest that differences across breeds in gene expression of the male gonads are larger than in other endocrine tissues in the pig. Nevertheless, the strong presence of breed biased genes in the male gonads cannot be explained solely by changes in spermatogenesis nor by differences in the reproductive tract development. PMID:19239697

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii. PMID:26824923

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii. PMID:26824923

A combination of 454 pyrosequencing and Sanger sequencing was used to sample and characterize the transcriptome of the entomopathogenic oomycete Lagenidium giganteum. More than 50,000 high-throughput reads were annotated through homology searches. Several selected reads served as seeds for the amplification and sequencing of full-length transcripts. Phylogenetic analyses inferred from full-length cellulose synthase alignments revealed that L giganteum is nested within the peronosporalean galaxy and as such appears to have evolved from a phytopathogenic ancestor. In agreement with the phylogeny reconstructions, full-length L. giganteum oomycete effector orthologs, corresponding to the cellulose-binding elicitor lectin (CBEL), crinkler (CRN), and elicitin proteins, were characterized by domain organizations similar to those of pathogenicity factors of plant-pathogenic oomycetes. Importantly, the L. giganteum effectors provide a basis for detailing the roles of canonical CRN, CBEL, and elicitin proteins in the infectious process of an oomycete known principally as an animal pathogen. Finally, phylogenetic analyses and genome mining identified members of glycoside hydrolase family 5 subfamily 27 (GH5_27) as putative virulence factors active on the host insect cuticle, based in part on the fact that GH5_27 genes are shared by entomopathogenic oomycetes and fungi but are underrepresented in nonentomopathogenic genomes. The genomic resources gathered from the L. giganteum transcriptomeanalysis strongly suggest that filamentous entomopathogens (oomycetes and fungi) exhibit convergent evolution: they have evolved independently from plant-associated microbes, have retained genes indicative of plant associations, and may share similar cores of virulence factors, such as GH5_27 enzymes, that are absent from the genomes of their plant-pathogenic relatives. PMID:25107973

White birch (Betula papyrifera) is a dominant tree species of the Boreal Forest. Recent studies have shown that it is fairly resistant to heavy metal contamination, specifically to nickel. Knowledge of regulation of genes associated with metal resistance in higher plants is very sketchy. Availability and annotation of the dwarf birch (B. nana) enables the use of high throughout sequencing approaches to understanding responses to environmental challenges in other Betula species such as B. papyrifera. The main objectives of this study are to 1) develop and characterize the B. papyrifera transcriptome, 2) assess gene expression dynamics of B. papyrifera in response to nickel stress, and 3) describe gene function based on ontology. Nickel resistant and susceptible genotypes were selected and used for transcriptomeanalysis. A total of 208,058 trinity genes were identified and were assembled to 275,545 total trinity transcripts. The transcripts were mapped to protein sequences and based on best match; we annotated the B. papyrifera genes and assigned gene ontology. In total, 215,700 transcripts were annotated and were compared to the published B. nana genome. Overall, a genomic match for 61% transcripts with the reference genome was found. Expression profiles were generated and 62,587 genes were found to be significantly differentially expressed among the nickel resistant, susceptible, and untreated libraries. The main nickel resistance mechanism in B. papyrifera is a downregulation of genes associated with translation (in ribosome), binding, and transporter activities. Five candidate genes associated to nickel resistance were identified. They include Glutathione S–transferase, thioredoxin family protein, putative transmembrane protein and two Nramp transporters. These genes could be useful for genetic engineering of birch trees. PMID:27082755

Salvia miltiorrhiza Bunge, a perennial plant of Lamiaceae, accumulates abietane-type diterpenoids of tanshinones in root, which have been used as traditional Chinese medicine to treat neuroasthenic insomnia and cardiovascular diseases. However, to date the biosynthetic pathway of tanshinones is only partially elucidated and the mechanism for their root-specific accumulation remains unknown. To identify enzymes and transcriptional regulators involved in the biosynthesis of tanshinones, we conducted transcriptome profiling of S. miltiorrhiza root and leaf tissues using the 454 GS-FLX pyrosequencing platform, which generated 550,546 and 525,292 reads, respectively. RNA sequencing reads were assembled and clustered into 64,139 unigenes (29,883 isotigs and 34,256 singletons). NCBI non-redundant protein databases (NR) and Swiss-Prot database searches anchored 32,096 unigenes (50%) with functional annotations based on sequence similarities. Further assignments with Gene Ontology (GO) terms and KEGG biochemical pathways identified 168 unigenes referring to the terpenoid backbone biosynthesis (including 144 MEP and MVA pathway genes and 24 terpene synthases). Comparative analysis of the transcriptomes identified 2,863 unigenes that were highly expressed in roots, including those encoding enzymes of early steps of tanshinone biosynthetic pathway, such as copalyl diphosphate synthase (SmCPS), kaurene synthase-like (SmKSL) and CYP76AH1. Other differentially expressed unigenes predicted to be related to tanshinone biosynthesis fall into cytochrome P450 monooxygenases, dehydrogenases and reductases, as well as regulatory factors. In addition, 21 P450 genes were selectively confirmed by real-time PCR. Thus we have generated a large unigene dataset which provides a valuable resource for further investigation of the radix development and biosynthesis of tanshinones. PMID:24260395

Jiyan-1 puffer, the F1 hybrid of Takifugu rubripes and Takifugu flavidus, displays obvious heterosis in the growth performance, flavor and stress tolerance. In the present study, comparative analysis for the transcriptomes of T. rubripes, T. flavidus and Jiyan-1 was performed aiming to reveal the possible mechanisms of heterosis in pufferfish. Whole transcriptomes were sequenced using the SOLiD4 platform, and a total of 44,305 transcripts corresponding to 18,164 genes were identified collectively. A total of 14,148 transcripts were differentially expressed. By comparing the gene expression patterns of the three samples, the coexistence of overdominance, dominance, underdominance and additivity was observed in the gene action modes of Jiyan-1. There were 2,237 transcripts in the intersection of the differentially expressed transcripts from Jiyan-1 versus T. rubripes and Jiyan-1 versus T. flavidus, among which 213 transcripts were also in the T. rubripes versus T. flavidus. The potential functions of the remaining 2,024 transcripts were mainly associated with metabolic process, nucleotide binding and catalytic activity. The enrichment results indicated metabolism was the most activated biological function in the heterosis. In addition, 35 KEGG pathways were retrieved as affiliated with more than three differentially expressed transcripts and 8,579 potentially novel transcript isoforms were identified for Jiyan-1. The present study revealed the coexistence of multiple gene actions in the hybrid puffer, indicated the importance of metabolism, ion binding function and kinase activity, as well as provided a list of candidate genes and pathways for heterosis. It could be helpful for the better understanding of the determination and regulation mechanisms of heterosis. PMID:23520511

Next-generation sequencing RNA-Seq technology is a powerful tool that creates new possibilities for whole-transcriptomeanalysis. In our study, the RNA-Seq method was applied to analyze global changes in transcriptome from muscle tissue (m. semimembranosus) in two pig breeds (Pietrain and Polish Landrace, PL). The breeds differ in terms of muscularity, growth rate and reproduction traits. Using three different approaches (deseq, cufflinks and edger) and taking into account the most restrictive criteria, 35 genes differentially expressed between Pietrain and PL pigs were identified. In both breeds, the most abundant were transcripts encoding ribosomal and cytoskeletal proteins (TPM3, TCAP, TMOD4, TPM2, TNNC1) and calcium-binding proteins involved in muscle contraction, calcium-mediated signaling or cation transport (CASQ1, MLC2V, SLC25A4, MYL3). In PL pigs, we identified up-regulation of several genes that play crucial roles in reproduction: female gamete generation (BDP1, PTPN21, USP9X), fertilization (EGFR) and embryonic development (CPEB4). In the Pietrain breed, only seven genes were over-expressed (CISH, SPP1, TUBA8, ATP6V1C2, IGKC, predicted LOC100510960 and LOC100626400), and they play important roles in, for example, negative regulation of apoptosis, immune response, cell-cell signaling, cell growth and migration as well as the metabolic process. The functions of the majority of selected genes were consistent with phenotypic variation in investigated breeds; thus, we proposed a new panel of candidate genes that can be associated with economically important pig traits. PMID:24961663

Introduction The potato (Solanum tuberosum) cultivar ‘Xin Daping’ is tetraploid with white skin and white flesh, while the cultivar ‘Hei Meiren’ is also tetraploid with purple skin and purple flesh. Comparative transcriptomeanalysis of white and purple cultivars was carried out using high-throughput RNA sequencing in order to further understand the mechanism of anthocyanin biosynthesis in potato. Methods and Results By aligning transcript reads to the recently published diploid potato genome and de novo assembly, 209 million paired-end Illumina RNA-seq reads from these tetraploid cultivars were assembled on to 60,930 transcripts, of which 27,754 (45.55%) are novel transcripts and 9393 alternative transcripts. Using a comparison of the RNA-sequence datasets, multiple versions of the genes encoding anthocyanin biosynthetic steps and regulatory transcription factors were identified. Other novel genes potentially involved in anthocyanin biosynthesis in potato tubers were also discovered. Real-time qPCR validation of candidate genes revealed good correlation with the transcriptome data. SNPs (Single Nucleotide Polymorphism) and indels were predicted and validated for the transcription factors MYB AN1 and bHLH1 and the biosynthetic gene anthocyanidin 3-O-glucosyltransferase (UFGT). Conclusions These results contribute to our understanding of the molecular mechanism of white and purple potato development, by identifying differential responses of biosynthetic gene family members together with the variation in structural genes and transcription factors in this highly heterozygous crop. This provides an excellent platform and resource for future genetic and functional genomic research. PMID:26053878

The widespread planting of genetically engineered cotton producing the Cry1Ac toxin has led to significantly reduced pesticide applications since 1997. However, consequently, the number of green mirid bugs (GMB), Apolygus lucorum (Meyer-Dür) has increased. So far the GMB, instead of the cotton bollworm Helicoverpa armigera (Hübner), has become the major pest in the transgenic Bt cotton field and has influenced cotton yield. Disproportionately, only a few studies on GMB at molecular level have been reported. Libraries from both third instar nymphs and adults were sequenced using Illumina technology, producing more than 106 million short reads and assembled into 63 029 unigenes of mean length 597 nt and N50 813 nt, ranging from 300 nt to 9771 nt. BLASTx analysis against Nr, Swissprot, GO and COG was performed to annotate these unigenes. As a result, 26 478 unigenes (42.01%) matched to known proteins and 107 immune-related, 320 digestive-related and 53 metamorphosis-related genes were detected in these annotated unigenes. Additionally, we profiled gene expression using mapping based differentially expressed genes (DEGs) strategy between the two developmental stages: nymph and adult. The results demonstrated that thousands of genes were significantly differentially expressed at different developmental stages. The transcriptome and gene expression data provided comprehensive and global gene resources of GMB. This transcriptome would improve our understanding of the molecular mechanisms of various underlying biological characteristics, including development, digestion and immunity in GMB. Therefore, these findings could help elucidate the intrinsic factors of the GMB resurgence, offering novel pest management targets for future transgenic cotton breeding. PMID:25408247

Background Grass carp (Ctenopharyngodon idella) is one of the most economically important freshwater fish, but its production is often affected by diseases that cause serious economic losses. To date, no good breeding varieties have been obtained using the oriented cultivation technique. The ability to identify disease resistance genes in grass carp is important to cultivate disease-resistant varieties of grass carp. Results In this study, we constructed a non-normalized cDNA library of head kidney in grass carp, and, after clustering and assembly, we obtained 3,027 high-quality unigenes. Solexa sequencing was used to generate sequence tags from the transcriptomes of the head kidney in grass carp before and after grass carp reovirus (GCRV) infection. After processing, we obtained 22,144 tags that were differentially expressed by more than 2-fold between the uninfected and infected groups. 679 of the differentially expressed tags (3.1%) mapped to 483 of the unigenes (16.0%). The up-regulated and down-regulated unigenes were annotated using gene ontology terms; 16 were annotated as immune-related and 42 were of unknown function having no matches to any of the sequences in the databases that were used in the similarity searches. Semi-quantitative RT-PCR revealed four unknown unigenes that showed significant responses to the viral infection. Based on domain structure predictions, one of these sequences was found to encode a protein that contained two transmembrane domains and, therefore, may be a transmembrane protein. Here, we proposed that this novel unigene may encode a virus receptor or a protein that mediates the immune signalling pathway at the cell surface. Conclusion This study enriches the molecular basis data of grass carp and further confirms that, based on fish tissue-specific EST databases, transcriptomeanalysis is an effective route to discover novel functional genes. PMID:22776770

The crucian carp is an important aquaculture species and a potential model to study genome evolution and physiological adaptation. However, so far the genomics and transcriptomics data available for this species are still scarce. We performed de novo transcriptome sequencing of four cDNA libraries representing brain, muscle, liver and kidney tissues respectively, each with six specimens. The removal of low quality reads resulted in 2.62 million raw reads, which were assembled as 127,711 unigenes, including 84,867 isotigs and 42,844 singletons. A total of 22,273 unigenes were found with significant matches to 14,449 unique proteins. Around14,398 unigenes were assigned with at least one Gene Ontology (GO) category in 84,876 total assignments, and 6,382 unigenes were found in 237 predicted KEGG pathways. The gene expression analysis revealed more genes expressed in brain, more up-regulated genes in muscle and more down-regulated genes in liver as compared with gene expression profiles of other tissues. In addition, 23 enzymes in the glycolysis/gluconeogenesis pathway were recovered. Importantly, we identified 5,784 high-quality putative SNP and 11,295 microsatellite markers which include 5,364 microsatellites with flanking sequences ≥50 bp. This study produced the most comprehensive genomic resources that have been derived from crucian carp, including thousands of genetic markers, which will not only lay a foundation for further studies on polyploidy origin and anoxic survival but will also facilitate selective breeding of this important aquaculture species. PMID:23630630

White birch (Betula papyrifera) is a dominant tree species of the Boreal Forest. Recent studies have shown that it is fairly resistant to heavy metal contamination, specifically to nickel. Knowledge of regulation of genes associated with metal resistance in higher plants is very sketchy. Availability and annotation of the dwarf birch (B. nana) enables the use of high throughout sequencing approaches to understanding responses to environmental challenges in other Betula species such as B. papyrifera. The main objectives of this study are to 1) develop and characterize the B. papyrifera transcriptome, 2) assess gene expression dynamics of B. papyrifera in response to nickel stress, and 3) describe gene function based on ontology. Nickel resistant and susceptible genotypes were selected and used for transcriptomeanalysis. A total of 208,058 trinity genes were identified and were assembled to 275,545 total trinity transcripts. The transcripts were mapped to protein sequences and based on best match; we annotated the B. papyrifera genes and assigned gene ontology. In total, 215,700 transcripts were annotated and were compared to the published B. nana genome. Overall, a genomic match for 61% transcripts with the reference genome was found. Expression profiles were generated and 62,587 genes were found to be significantly differentially expressed among the nickel resistant, susceptible, and untreated libraries. The main nickel resistance mechanism in B. papyrifera is a downregulation of genes associated with translation (in ribosome), binding, and transporter activities. Five candidate genes associated to nickel resistance were identified. They include Glutathione S-transferase, thioredoxin family protein, putative transmembrane protein and two Nramp transporters. These genes could be useful for genetic engineering of birch trees. PMID:27082755

In this paper, we apply the detrended fluctuation analysis (DFA), local scaling detrended fluctuation analysis (LSDFA), and detrended cross-correlation analysis (DCCA) to investigate correlations of several stock markets. DFA method is for the detection of long-range correlations used in time series. LSDFA method is to show more local properties by using local scale exponents. DCCA method is a developed method to quantify the cross-correlation of two non-stationary time series. We report the results of auto-correlation and cross-correlation behaviors in three western countries and three Chinese stock markets in periods 2004-2006 (before the global financial crisis), 2007-2009 (during the global financial crisis), and 2010-2012 (after the global financial crisis) by using DFA, LSDFA, and DCCA method. The findings are that correlations of stocks are influenced by the economic systems of different countries and the financial crisis. The results indicate that there are stronger auto-correlations in Chinese stocks than western stocks in any period and stronger auto-correlations after the global financial crisis for every stock except Shen Cheng; The LSDFA shows more comprehensive and detailed features than traditional DFA method and the integration of China and the world in economy after the global financial crisis; When it turns to cross-correlations, it shows different properties for six stock markets, while for three Chinese stocks, it reaches the weakest cross-correlations during the global financial crisis.

There is enormous interest in developing renewable sources of liquid fuels because of depletion of fossil fuel reserves, dependence on foreign sources, and increasing atmospheric CO2 levels. Algae produce neutral lipids that are readily converted into liquid fuels such as biodiesel or JP-8 equivalent, and are attractive sources because they are far more productive than plants (yielding 10 -100’s of time more lipid per land area), and can be grown on non-cultivatable land with non-potable (brackish or salt) water sources. Unicellular algae known as diatoms were the most thoroughly characterized species in the National Renewable Energy Laboratory’s Aquatic Species Program, whose goal was to develop microalgae as renewable fuel sources. Lipid accumulation in microalgae is generally induced by nutrient limitation, which involves a change in environmental conditions. Intrinsic variability in cellular response to environmental changes prevents a high degree of control over the process. Nutrient limitation also inhibits biomass accumulation; therefore a tradeoff between high biomass and lipid production occurs. The goal of this project was to develop metabolic engineering approaches for diatoms to enable induction of lipid accumulation by controllable manipulation of intracellular processes rather than from external environmental conditions, and to manipulate carbon partitioning within the cell between lipid and carbohydrate synthesis to enable both abundant biomass and lipid accumulation. There were two specific objectives for this project; Objective 1:To perform comparative transcriptomicanalysis in T. pseudonana and C. cryptica of lipid accumulation resulting from silicon and nitrogen limitation, to identify common and key regulatory steps involved in controlling lipid accumulation and carbon partitioning; and Objective 2: To metabolically engineer the cell to alter carbon partitioning to either trigger lipid induction without the need for nutrient

Characterization of the transcriptomic response to infection is an effective approach to understanding the immune mechanisms. Herein we challenged grass carp (Ctenopharyngodon idella) with grass carp reovirus (GCRV) and sequenced four cDNA libraries obtained from head-kidney and spleen by using Illumina Miseq. As a result, we gained a total of 21.52 Gb clean data with 107.96 million reads, and de novo assembled 55,199 unigenes with an average length of 1,470 bp. Comparative transcriptomeanalysis reveals that 217 unigenes are differentially expressed (fold-change of at least 4) between resistant and susceptible fish in both head-kidney and spleen, and of which 36 unigenes were validated by RT-qPCR experiment. The expression profile of immune-related genes demonstrates that the immune response of spleen is more intense than that of head-kidney. Remarkably, 11,811 unigenes contain multiple transcripts, of which 322 unigenes possess notably differentially expressed transcripts between the four transcriptomic datasets. Furthermore, the splicing transcripts of IL-12p40 and IL-1R1 are firstly found to play diverse roles in the antiviral response of fishes. This study provides a complete transcriptome dataset of C. idella, which is valuable for the studies of immune complexity and, moreover, throws light on the regulatory role of AS in antiviral immunity. PMID:26248502

Background The brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most serious rice insect pests in Asia, can quickly overcome rice resistance by evolving new virulent populations. The insect fat body plays essential roles in the life cycles of insects and in plant-insect interactions. However, whether differences in fat body transcriptomes exist between insect populations with different virulence levels and whether the transcriptomic differences are related to insect virulence remain largely unknown. Methodology/Principal Findings In this study, we performed transcriptome-wide analyses on the fat bodies of two BPH populations with different virulence levels in rice. The populations were derived from rice variety TN1 (TN1 population) and Mudgo (M population). In total, 33,776 and 32,332 unigenes from the fat bodies of TN1 and M populations, respectively, were generated using Illumina technology. Gene ontology annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology classifications indicated that genes related to metabolism and immunity were significantly active in the fat bodies. In addition, a total of 339 unigenes showed homology to genes of yeast-like symbionts (YLSs) from 12 genera and endosymbiotic bacteria Wolbachia. A comparative analysis of the two transcriptomes generated 7,860 differentially expressed genes. GO annotations and enrichment analysis of KEGG pathways indicated these differentially expressed transcripts might be involved in metabolism and immunity. Finally, 105 differentially expressed genes from YLSs and Wolbachia were identified, genes which might be associated with the formation of different virulent populations. Conclusions/Significance This study was the first to compare the fat-body transcriptomes of two BPH populations having different virulence traits and to find genes that may be related to this difference. Our findings provide a molecular resource for future investigations of fat bodies and will be useful

Background The Trachinotus ovatus (Teleostei, Carangidae) is an economically important marine fish species in the world. However, the lack of genomic information regarding this species limits our understanding of the genetics and biological mechanisms in Trachinotus ovatus. In this study, high throughput transcriptome sequencing was used to obtain comprehensive genomic information in Trachinotus ovatus. Principal Findings Transcriptome sequencing was performed by using Illumina paired-end sequencing technology. The 98,534,862 high quality reads were yielded, and were de novo assembled into 156,094 unigenes with an average sequence length of 1179 bp. Transcriptome annotation revealed that 75,586 and 67,923 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 67,923 unigenes were grouped into 25 Cluster of Orthologous Groups (COG) functional categories, 37,976 unigenes were clustered into 61 Gene Ontology (GO) terms, and 38,172 unigenes were assigned to 275 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Based on the transcriptome dataset, a large number of unigenes associated with reproduction, growth and immunity were identified. Furthermore, a total number of 38,794 simple sequence repeats (SSRs) were discovered and 16 polymorphic loci were characterized in Trachinotus ovatus. Conclusion/Significance The present study is the first transcriptomeanalysis of a fish species belonging to the genus Trachinotus and provides a valuable genomic resource for novel gene discovery, gene expression and regulation studies, and the identification of genetic markers in Trachinotus ovatus and the other fish of the genus Trachinotus. PMID:25303650

Environmental salinity creates a key barrier to limit the distribution of most aquatic organisms. Adaptation to osmotic fluctuation is believed to be a factor facilitating species diversification. Adaptive evolution often involves beneficial mutations at more than one locus. Bivalves hold great interest, with numerous species living in waters, as osmoconformers, who maintain the osmotic pressure balance mostly by free amino acids. In this study, 107,076,589 reads from two groups of Crassostrea hongkongensis were produced and the assembled into 130,629 contigs. Transcripts putatively involved in stress-response, innate immunity and cell processes were identified according to Gene ontology and KEGG pathway analyses. Comparing with the transcriptome of C. gigas to characterize the diversity of transcripts between species with osmotic divergence, we identified 182,806 high-quality single nucleotide polymorphisms (SNPs) for C. hongkongensis, and 196,779 SNPs for C. gigas. Comparison of 11,602 pairs of putative orthologs allowed for identification of 14 protein-coding genes that experienced strong positive selection (Ka/Ks>1). In addition, 45 genes that may show signs of moderate positive selection (1≥Ka/Ks>0.5) were also identified. Based on Ks ratios and divergence time between the two species published previously, we estimated a neutral transcriptome-wide substitution mutation rate of 1.39×10−9 per site per year. Several genes were differentially expressed across the control and treated groups of each species. This is the first time to sequence the transcriptome of C. hongkongensis and provide the most comprehensive transcriptomic resource available for it. The increasing amount of transcriptome data on Crassostrea provides an excellent resource for phylogenetic analysis. A large number of SNPs identified in this work are expected to provide valuable resources for future marker and genotyping assay development. The analysis of natural selection provides an

Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomicanalysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning

Bipolaris zeicola is a fungal pathogen that causes Northern corn leaf spot (NCLS), which is a serious foliar disease in maize and one of the most significant pathogens affecting global food security. Here, we report a genome-wide transcriptional profile analysis using next-generation sequencing (NGS) of maize leaf development after inoculation with B. zeicola. We performed High-Throughput Digital Gene Expression analysis to identify differentially expressed genes (DEGs) in resistant inbred Mo17 lines after infection with B. zeicola at four successive disease development stages--CP (contact period), PP (penetration period), IP (incubation period), and DP (disease period); the expression of the genes was compared with those in a CK (mock-treatment) control. In addition, a sensitive maize line (Zheng58) was used for the comparisons with the Mo17. Among all tested genes, 466 differentially expressed genes were identified in all libraries, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these genes suggested that they are involved in many biological processes related to systemic symptom development, such as plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis and photosynthesis. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in fungal-infected plants. This information will help in furthering our understanding of the detailed mechanisms of plant responses to fungal infection. PMID:25781606

Objective Identification of colorectal cancer (CRC) metastasis genes is one of the most important issues in CRC research. For the purpose of mining CRC metastasis-associated genes, an integrated analysis of microarray data was presented, by combined with evidence acquired from comparative genomic hybridization (CGH) data. Methods Gene expression profile data of CRC samples were obtained at Gene Expression Omnibus (GEO) website. The 15 important chromosomal aberration sites detected by using CGH technology were used for integrated genomic and transcriptomicanalysis. Significant Analysis of Microarray (SAM) was used to detect significantly differentially expressed genes across the whole genome. The overlapping genes were selected in their corresponding chromosomal aberration regions, and analyzed by using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Finally, SVM-T-RFE gene selection algorithm was applied to identify metastasis-associated genes in CRC. Results A minimum gene set was obtained with the minimum number [14] of genes, and the highest classification accuracy (100%) in both PRI and META datasets. A fraction of selected genes are associated with CRC or its metastasis. Conclusions Our results demonstrated that integration analysis is an effective strategy for mining cancer-associated genes. PMID:24385689

Phytoplasmas are plant pathogenic bacteria that have no cell wall and are responsible for major crop losses throughout the world. Phytoplasma-infected plants show a variety of symptoms and the mechanisms they use to physiologically alter the host plants are of considerable interest, but poorly understood. In this study we undertook a detailed analysis of Paulownia infected by Paulownia witches’-broom (PaWB) Phytoplasma using high-throughput mRNA sequencing (RNA-Seq) and digital gene expression (DGE). RNA-Seq analysis identified 74,831 unigenes, which were subsequently used as reference sequences for DGE analysis of diseased and healthy Paulownia in field grown and tissue cultured plants. Our study revealed that dramatic changes occurred in the gene expression profile of Paulownia after PaWB Phytoplasma infection. Genes encoding key enzymes in cytokinin biosynthesis, such as isopentenyl diphosphate isomerase and isopentenyltransferase, were significantly induced in the infected Paulownia. Genes involved in cell wall biosynthesis and degradation were largely up-regulated and genes related to photosynthesis were down-regulated after PaWB Phytoplasma infection. Our systematic analysis provides comprehensive transcriptomic data about plants infected by Phytoplasma. This information will help further our understanding of the detailed interaction mechanisms between plants and Phytoplasma. PMID:24130859

Bipolaris zeicola is a fungal pathogen that causes Northern corn leaf spot (NCLS), which is a serious foliar disease in maize and one of the most significant pathogens affecting global food security. Here, we report a genome-wide transcriptional profile analysis using next-generation sequencing (NGS) of maize leaf development after inoculation with B. zeicola. We performed High-Throughput Digital Gene Expression analysis to identify differentially expressed genes (DEGs) in resistant inbred Mo17 lines after infection with B. zeicola at four successive disease development stages—CP (contact period), PP (penetration period), IP (incubation period), and DP (disease period); the expression of the genes was compared with those in a CK (mock-treatment) control. In addition, a sensitive maize line (Zheng58) was used for the comparisons with the Mo17. Among all tested genes, 466 differentially expressed genes were identified in all libraries, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these genes suggested that they are involved in many biological processes related to systemic symptom development, such as plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis and photosynthesis. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in fungal-infected plants. This information will help in furthering our understanding of the detailed mechanisms of plant responses to fungal infection. PMID:25781606

Grapefruit (Citrus pardisi) is a popular citrus fruit that is a cross between a sweet orange and pummelo. This research article focuses on an in silico approach for comparative analysis of C. paradisi green flavedo (GF) and ethylene treated flavedo (ETF) transcriptome data. Our pathway analysis provides comprehensive information of genes playing significant role in different stages of ripening in fruit. De novo assembly was carried out using six different assemblers namely GS assembler, SeqMan NGEN, Velvet/Oases, CLC, iAssembler and Cortex followed by subsequent meta-assembly, annotation and pathway analysis. We conclude that de novo transcriptome assembly using meta-assembly approach is used to increase assembly quality in comparison to single assembler. PMID:24477585

High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism's transcriptome. Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at

High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism's transcriptome. Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at

Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS transcription factors (Hsfs). The role of Hsfs and Hsps in HS response in tomato was initially examined by transcriptomeanalysis using the massive analysis of cDNA ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points towards two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato. PMID:25124075

The central nervous system (CNS) is composed of hundreds of distinct cell types, each expressing different subsets of genes from the genome. High throughput gene expression analysis of the CNS from patients and controls is a common method to screen for potentially pathological molecular mechanisms of psychiatric disease. One mechanism by which gene expression might be seen to vary across samples would be alterations in the cellular composition of the tissue. While the expressions of gene ‘markers’ for each cell type can provide certain information of cellularity, for many rare cell types markers are not well characterized. Moreover, if only small sets of markers are known, any substantial variation of a marker’s expression pattern due to experiment conditions would result in poor sensitivity and specificity. Here, our proposed method combines prior information from mice cell-specific transcriptome profiling experiments with co-expression network analysis, to select large sets of potential cell type-specific gene markers in a systematic and unbiased manner. The method is efficient and robust, and identifies sufficient markers for further cellularity analysis. We then employ the markers to analytically detect changing cellular composition in human brain. Application of our method to temporal human brain microarray data successfully detects changes in cellularity over time that roughly correspond to known epochs of human brain development. Furthermore, application of our method to human brain samples with the neurodevelopmental disorder of autism supports the interpretation that the changes in astrocytes and neurons might contribute to the disorder. PMID:25340014

Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptomeanalysis of the fungal aging model Podospora anserina. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wild-type transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model P. anserina which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations. PMID:24376646

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptomeanalysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack. PMID:25506299

Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often life-threatening. Resistance of C. albicans to antifungal agents and limited antifungal agents has potentially serious implications for management of infections. As a famous multiherb prescription in China, Huanglian Jiedu Decoction (HLJJD, Orengedokuto in Japan) is efficient against Trichophyton mentagrophytes and C. albicans. But the antifungal mechanism of HLJDD remains unclear. In this study, by using RNA-seq technique, we performed a transcriptomicsanalysis of gene expression changes for C. albicans under the treatment of HLJDD. A total of 6057 predicted protein-encoding genes were identified. By gene expression analysis, we obtained a total of 735 differentially expressed genes (DEGs), including 700 upregulated genes and 35 downregulated genes. Genes encoding multidrug transporters such as ABC transporter and MFS transporter were identified to be significantly upregulated. Meanwhile, by pathway enrichment analysis, we identified 26 significant pathways, in which pathways of DNA replication and transporter activity were mainly involved. These results might provide insights for the inhibition mechanism of HLJDD against C. albicans. PMID:27143984

Sufficient light is essential for the growth and physiological functions of photosynthetic organisms, but prolonged exposure to high light (HL) stress can cause cellular damage and ultimately result in the death of these organisms. Synechococcus sp. PCC 7002 (hereafter Synechococcus 7002) is a unicellular cyanobacterium with exceptional tolerance to HL intensities. However, the molecular mechanisms involved in HL response by Synechococcus 7002 are not well understood. Here, an integrated RNA sequencing transcriptomic and quantitative proteomic analysis was performed to investigate the cellular response to HL in Synechococcus 7002. A total of 526 transcripts and 233 proteins were identified to be differentially regulated under HL stress. Data analysis revealed major changes in mRNAs and proteins involved in the photosynthesis pathways, resistance to light-induced damage, DNA replication and repair, and energy metabolism. A set of differentially expressed mRNAs and proteins were validated by quantitative RT-PCR and Western blot, respectively. Twelve genes differentially regulated under HL stress were selected for knockout generation and growth analysis of these mutants led to the identification of key genes involved in the response of HL in Synechococcus 7002. Taken altogether, this study established a model for global response mechanisms to HL in Synechococcus 7002 and may be valuable for further studies addressing HL resistance in photosynthetic organisms. PMID:25681118

Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often life-threatening. Resistance of C. albicans to antifungal agents and limited antifungal agents has potentially serious implications for management of infections. As a famous multiherb prescription in China, Huanglian Jiedu Decoction (HLJJD, Orengedokuto in Japan) is efficient against Trichophyton mentagrophytes and C. albicans. But the antifungal mechanism of HLJDD remains unclear. In this study, by using RNA-seq technique, we performed a transcriptomicsanalysis of gene expression changes for C. albicans under the treatment of HLJDD. A total of 6057 predicted protein-encoding genes were identified. By gene expression analysis, we obtained a total of 735 differentially expressed genes (DEGs), including 700 upregulated genes and 35 downregulated genes. Genes encoding multidrug transporters such as ABC transporter and MFS transporter were identified to be significantly upregulated. Meanwhile, by pathway enrichment analysis, we identified 26 significant pathways, in which pathways of DNA replication and transporter activity were mainly involved. These results might provide insights for the inhibition mechanism of HLJDD against C. albicans. PMID:27143984

To comprehensively reflect the roles of Rpl11 on the transcriptome of zebrafish model of Diamond-Blackfan Anemia (DBA), we performed whole-genome transcriptome sequencing on the Illumina Hi-Seq 2000 sequencing platform. Two different transcriptomes of zebrafish Rpl11-deficient and control Morpholino (Mo) embryos were collected and analyzed. The experimental design and methods, including sample preparation, RNA-Seq data evaluation and treatment, were described in details so that representative high-throughput sequencing data were acquired for assessing the actual impacts of Rpl11 on zebrafish embryos. We provided the accession number GSE51326 for easy access to the database. PMID:26484089

Kelps, brown algae of the order Laminariales, dominate rocky shores and form huge kelp beds which provide habitat and nurseries for various marine organisms. Whereas the basic physiological and ecophysiological characteristics of kelps are well studied, the molecular processes underlying acclimation to different light and temperature conditions are still poorly understood. Therefore we investigated the molecular mechanisms underlying the physiological acclimation to light and temperature stress. Sporophytes of S. latissima were exposed to combinations of light intensities and temperatures, and microarray hybridizations were performed to determine changes in gene expression patterns. This first large-scaletranscriptomic study of a kelp species shows that S. latissima responds to temperature and light stress with a multitude of transcriptional changes: up to 32% of genes showed an altered expression after the exposure experiments. High temperature had stronger effects on gene expression in S. latissima than low temperature, reflected by the higher number of temperature-responsive genes. We gained insights into underlying molecular processes of acclimation, which includes adjustment of the primary metabolism as well as induction of several ROS scavengers and a sophisticated regulation of Hsps. We show that S. latissima, as a cold adapted species, must make stronger efforts for acclimating to high than to low temperatures. The strongest response was caused by the combination of high temperatures with high light intensities, which proved most harmful for the alga. PMID:22937172

This report describes an integrated study on identification of potential markers for gastric cancer in patients’ cancer tissues and sera based on: (i) genome-scaletranscriptomic analyses of 80 paired gastric cancer/reference tissues and (ii) computational prediction of blood-secretory proteins supported by experimental validation. Our findings show that: (i) 715 and 150 genes exhibit significantly differential expressions in all cancers and early-stage cancers versus reference tissues, respectively; and a substantial percentage of the alteration is found to be influenced by age and/or by gender; (ii) 21 co-expressed gene clusters have been identified, some of which are specific to certain subtypes or stages of the cancer; (iii) the top-ranked gene signatures give better than 94% classification accuracy between cancer and the reference tissues, some of which are gender-specific; and (iv) 136 of the differentially expressed genes were predicted to have their proteins secreted into blood, 81 of which were detected experimentally in the sera of 13 validation samples and 29 found to have differential abundances in the sera of cancer patients versus controls. Overall, the novel information obtained in this study has led to identification of promising diagnostic markers for gastric cancer and can benefit further analyses of the key (early) abnormalities during its development. PMID:20965966

Background Global warming predictions indicate that temperatures will increase by another 2-6°C by the end of this century. High temperature is a major abiotic stress limiting plant growth and productivity in many areas of the world. Switchgrass (Panicum virgatum L.) is a model herbaceous bioenergy crop, due to its rapid growth rate, reliable biomass yield, minimal requirements of water and nutrients, adaptability to grow on marginal lands and widespread distribution throughout North America. The effect of high temperature on switchgrass physiology, cell wall composition and biomass yields has been reported. However, there is void in the knowledge of the molecular responses to heat stress in switchgrass. Results We conducted long-term heat stress treatment (38°/30°C, day/night, for 50 days) in the switchgrass cultivar Alamo. A significant decrease in the plant height and total biomass was evident in the heat stressed plants compared to controls. Total RNA from control and heat stress samples were used for transcriptomeanalysis with switchgrass Affymetrix genechips. Following normalization and pre-processing, 5365 probesets were identified as differentially expressed using a 2-fold cutoff. Of these, 2233 probesets (2000 switchgrass unigenes) were up-regulated, and 3132 probesets (2809 unigenes) were down-regulated. Differential expression of 42 randomly selected genes from this list was validated using RT-PCR. Rice orthologs were retrieved for 78.7% of the heat stress responsive switchgrass probesets. Gene ontology (GOs) enrichment analysis using AgriGO program showed that genes related to ATPase regulator, chaperone binding, and protein folding was significantly up-regulated. GOs associated with protein modification, transcription, phosphorus and nitrogen metabolic processes, were significantly down-regulated by heat stress. Conclusions Plausible connections were identified between the identified GOs, physiological responses and heat response phenotype

Background Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here, we perform transcriptomeanalysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyze 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumors without known driver mutations. In addition, we observe exon skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations. PMID:25531467

Dongxiang wild rice (Oryza rufipogon Griff.) is the progenitor of cultivated rice (Oryza sativa L.), and is well known for its superior level of tolerance against cold, drought and diseases. To date, however, little is known about the salt-tolerant character of Dongxiang wild rice. To elucidate the molecular genetic mechanisms of salt-stress tolerance in Dongxiang wild rice, the Illumina HiSeq 2000 platform was used to analyze the transcriptome profiles of the leaves and roots at the seedling stage under salt stress compared with those under normal conditions. The analysis results for the sequencing data showed that 6,867 transcripts were differentially expressed in the leaves (2,216 up-regulated and 4,651 down-regulated) and 4,988 transcripts in the roots (3,105 up-regulated and 1,883 down-regulated). Among these differentially expressed genes, the detection of many transcription factor genes demonstrated that multiple regulatory pathways were involved in salt stress tolerance. In addition, the differentially expressed genes were compared with the previous RNA-Seq analysis of salt-stress responses in cultivated rice Nipponbare, indicating the possible specific molecular mechanisms of salt-stress responses for Dongxiang wild rice. A large number of the salt-inducible genes identified in this study were co-localized onto fine-mapped salt-tolerance-related quantitative trait loci, providing candidates for gene cloning and elucidation of molecular mechanisms responsible for salt-stress tolerance in rice. PMID:26752408

Withania somnifera is one of the most valuable medicinal plants synthesizing secondary metabolites known as withanolides. Despite pharmaceutical importance, limited information is available about the biosynthesis of withanolides. Chemo-profiling of leaf and root tissues of Withania suggest differences in the content and/or nature of withanolides in different chemotypes. To identify genes involved in chemotype and/or tissue-specific withanolide biosynthesis, we established transcriptomes of leaf and root tissues of distinct chemotypes. Genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis with their alternatively spliced forms and paralogous have been identified. Analysis suggests differential expression of large number genes among leaf and root tissues of different chemotypes. Study also identified differentially expressing transcripts encoding cytochrome P450s, glycosyltransferases, methyltransferases and transcription factors which might be involved in chemodiversity in Withania. Virus induced gene silencing of the sterol ∆7-reductase (WsDWF5) involved in the synthesis of 24-methylene cholesterol, withanolide backbone, suggests role of this enzyme in biosynthesis of withanolides. Information generated, in this study, provides a rich resource for functional analysis of withanolide-specific genes to elucidate chemotype- as well as tissue-specific withanolide biosynthesis. This genomic resource will also help in development of new tools for functional genomics and breeding in Withania. PMID:26688389

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis, and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomicanalysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future. PMID:26403200